Compositions and methods for treating bone defects

ABSTRACT

A bone graft composition includes a biologically-resorbable cement and a plurality of processed bone particles, where each of the bone particles have a shape configured to interconnect with adjacent bone particles. A method for treating a bone defect using the bone graft compositions includes providing the bone graft composition and administering an effective amount of the bone graft composition to a site of a bone defect in a subject. Kits including a biologically-resorbable cement powder and a plurality of processed bone particles are also provided.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/219,376, filed Aug. 21, 2011, which claimspriority from U.S. Provisional Application Ser. No. 61/377,262, filedAug. 26, 2010, the entire disclosures of which are incorporated hereinby this reference.

TECHNICAL FIELD

The presently-disclosed subject matter relates to compositions andmethods for treating bone defects and, more particularly, to bone graftcompositions where the mechanical properties, incorporation, andremodeling of a biologically-resorbable cement are improved byaugmenting the cement with processed bone particles.

BACKGROUND

Over 500,000 bone graft procedures are performed annually in the UnitedStates, and approximately 2.2 million are performed worldwide with anannual cost of nearly $2.5 billion. These bone graft procedures areroutinely performed to not only treat bone fractures and other bonedefects, but are also routinely performed to strengthen existing bonethat may be deteriorating. Typically, the bone material used for thesebone graft procedures is either autograft, which is derived from thepatient's own body, or allograft, which is derived from a geneticallydissimilar member of the same species. In some cases though, the graftmaterial can even be xenograft, which is taken from another species.

From a biological standpoint, autograft is the preferred type of graftmaterial and the type of material that is most commonly used in many ofthe orthopedic, maxillofacial, podiatric, and dental surgeries thatrequire bone graft procedures to be performed. Autograft bone materialsalso exhibit many of the preferred properties for treating a bonedefect, including the ability to produce new bone from transplantedliving cells and the ability to integrate with the bone tissue at thegraft site. Despite these advantages, however, an autograft procedureusually requires that additional surgery be performed on a subject toacquire the graft material, which can lead to complications, such asinflammation or infection. In addition, during these surgeries, only avery limited amount of bone can be collected. As such, allograft andxenograft materials have been developed that provide benefits in termsof the quantity of materials that can be obtained, but those materialsstill frequently have their own complications, such as diseasetransmission and graft failure, thus leaving researchers looking forbetter alternatives.

To that end, many additional types of bone graft compositions have beenrecently developed, including allograft-based, ceramic-based, andpolymer-based compositions. For example, U.S. Pat. No. 7,494,950describes implantable compositions containing a calcium salt-containingcomponent, optionally demineralized bone, and a plurality of discretefibers. For another example, U.S. Pat. No. 6,548,080 describes anapplication for a bone defect site that includes a partiallydemineralized cortical bone structure. As yet another example, U.S. Pat.No. 6,599,516 describes the inclusion of materials within a moldableceramic compound capable of hardening, with the specific goal ofallowing cellular access to the interior of the implanted material.Nevertheless, despite the many alternative bone graft compositionsavailable today, the currently-available alternative bone graftcompositions generally do not possess sufficient strength and are notrapidly or completely incorporated, remodeled, or resorbed by the bodyof a subject. Thus, they can not be considered as viable alternatives toprior autograft-, allograft-, or xenograft-based bone graft materials.Furthermore, currently-available bone graft compositions do notsufficiently address how certain concentrations or shapes of the boneparticles can be incorporated into a bone graft composition in a mannerthat changes the properties of the composition itself and increases thestrength, resorption rate, and rate of incorporation and remodeling ofthe implanted materials.

SUMMARY

This Summary describes several embodiments of the presently-disclosedsubject matter, and, in many cases, lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently-disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

The presently-disclosed subject matter includes bone graft compositions,methods of using the bone graft compositions to treat a bone defect, andkits comprising the components of the bone graft compositions. Inparticular, the presently-disclosed subject matter provides bone graftcompositions, methods of using those compositions, and kits comprisingthe components of the bone graft compositions, where the mechanicalproperties and incorporation of a biologically-resorbable cement isincreased by augmenting the cement with processed bone particles havingan interconnecting shape.

In some embodiments of the presently-disclosed subject matter, a bonegraft composition is provided that comprises a biologically-resorbablecement and a plurality of processed bone particles. In thesecompositions, each of the bone particles has an interconnecting shape(e.g., a dumbbell shape), such that each bone particle is capable ofinterconnecting with adjacent bone particles when it is included in thebone graft composition. In some embodiments, the interconnecting of theshaped bone particles increases the mechanical properties of thebiologically-resorbable cement. In some embodiments, the bone particlesare also configured to interlock with adjacent bone particles and/orconfigured to interdigitate with the surrounding cement such that theinterlocking and/or interdigitating of the bone particles furtherincreases the mechanical properties of the biologically-resorbablecement. In some embodiments, a cross-section of a portion of theprocessed bone particles is substantially round, elliptical, square,rectangular, or triangular in shape, such that the cement is furtherimproved in compression, shear, tension, and bending loading modes ascompared to a cement composition that does not include bone particles orone that includes randomly-shaped or randomly-oriented particles.

With respect to the biologically-resorbable cements utilized inaccordance with the presently-disclosed bone graft compositions, in someembodiments, the biologically-resorbable cements are comprised of acalcium-based cement. In some embodiments, the calcium-based cement is acalcium phosphate cement. In certain embodiments, the calcium-basedcement is a hydroxyapatite cement. In other embodiments, thecalcium-based cement is a calcium sulfate cement.

The processed bone particles of the presently-disclosed bone graftcompositions are typically combined with the cement at a concentrationof about 1 percent to about 50 percent by volume of the bone graftcomposition or, in some embodiments, at a concentration of about 1 toabout 15 percent by volume of the bone graft composition. In someembodiments of the presently-disclosed bone graft compositions, theprocessed bone particles are about 5 percent to about 90 percentdemineralized. In such embodiments, the processed bone particles aretypically comprised of cortical bone particles. In other embodiments ofthe presently-disclosed bone graft compositions, the bone particles arecomprised of cancellous bone particles. In further embodiments, the boneparticles include both cortical and cancellous bone.

Further, the processed bone particles of the presently-disclosed bonegraft compositions can, in some embodiments, be selected from autograftbone particles, allograft bone particles, xenograft bone particles, andcombinations thereof. In some embodiments, the bone graft compositionscan further include an antibiotic, an osteoinductive material, anosteogenic material, or both an osteoinductive and an osteogenicmaterial.

Still further provided, in some embodiments of the presently-disclosedsubject matter, are methods for treating a bone defect that make use ofthe bone graft compositions described herein. In some embodiments, amethod for treating a bone defect is provided that comprises the stepsof providing a bone graft composition of the presently-disclosed subjectmatter and administering an effective amount of the bone graftcomposition to a site of a bone defect in a subject. In someembodiments, the bone defect is a bone void, a fracture, or the site ofan intended bone fusion. Each of these bone defects are treated, in someembodiments, by filling the bone defect with a bone graft composition ofthe presently-disclosed subject matter.

In yet further embodiments of the presently-disclosed subject matter,kits are provided. In some embodiments, a kit is provided that includesa biologically-resorbable cement powder and a plurality of processedbone particles, where each of the processed bone particles has a shapeconfigured to interconnect with adjacent bone particles. In someembodiments of the kits, the biologically-resorbable cement and theprocessed bone particles are packed in separate vessels or are packagedtogether in a single vessel. In some embodiments, the bone particles arelyophilized. In this regard, in some embodiments, the kit furtherincludes water or another aqueous vehicle for adding to the cementpowder, the bone particles, or both the cement powder and the boneparticles. In some embodiments, the kit further comprises instructionsfor mixing the cement powder and the bone particles, and then combiningthat mixture with an aqueous vehicle such that a desired bone graftcomposition is produced.

Further advantages of the presently-disclosed subject matter will becomeevident to those of ordinary skill in the art after a study of thedescription, Figures, and non-limiting Examples in this document.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a perspective view of a dumbbell-shaped bone particle made inaccordance with the presently-disclosed subject matter;

FIG. 1B is a perspective view of another dumbbell-shaped bone particlemade in accordance with the presently-disclosed subject matter;

FIG. 1C is a perspective view of a further dumbbell-shaped bone particlemade in accordance with the presently-disclosed subject matter;

FIG. 1D is a side view of an elongated bone particle made in accordancewith the presently-disclosed subject matter;

FIG. 1E is a perspective view of yet another dumbbell-shaped boneparticle made in accordance with the presently-disclosed subject matter;

FIG. 1F is a side view of another elongated bone particle made inaccordance with the presently-disclosed subject matter;

FIG. 1G is a side view of a further elongated bone particle made inaccordance with the presently-disclosed subject matter;

FIG. 2 is a schematic diagram showing a plurality of dumbbell-shapedbone particles interconnected with one another;

FIG. 3 is a schematic diagram showing an experimental three-pointbending fixture used to apply a force (F) and assess the bendingstrength of a hardened bone graft composition (cylindrical specimen)made in accordance with the presently-disclosed subject matter;

FIG. 4 is a graph showing the results of a bending test performed with athree-point bending fixture as shown in FIG. 3 to analyze the bendingstrength of: a pure, calcium phosphate cement (CaP 100%) comprised oftetracalcium phosphate (TTCP); monocalcium phosphate (MCP), and calciumcarbonate; a bone graft composition of the presently-disclosed subjectmatter that includes a calcium phosphate cement comprised of TTCP, MCPand calcium carbonate and that includes 10 percent or 20 percent byvolume of processed bone particles having a shape as shown in FIG. 1A(Tr 10% and Tr 20%, respectively); and a bone graft composition thatincludes a calcium phosphate cement comprised of TTCP, MCP and calciumcarbonate and that includes 10 percent or 40 percent by volume ofnon-specially shaped bone particles (Alt 10% and Alt 40%, respectively);

FIG. 5 is a graph showing the results of a bending test performed with athree-point bending fixture as shown in FIG. 3 to analyze the bendingstrength of: a pure, commercial-grade calcium phosphate cement (CaP*100%) made from alpha-tricalcium phosphate powder; a bone graftcomposition of the presently-disclosed subject matter that includes acommercial-grade calcium phosphate cement made from alpha-tricalciumphosphate powder and that includes 10 percent by volume of processedbone particles having a shape as shown in FIG. 1A (Tr 10%); and a bonegraft composition that includes a commercial-grade calcium phosphatecement made from alpha-tricalcium phosphate powder and that includes 40percent by volume of non-specially shaped bone particles (Alt 40%);

FIG. 6 is a graph showing the results of a bending test performed with athree-point bending fixture as shown in FIG. 3 to analyze the bendingstrength of: a pure, commercial grade calcium sulfate cement (CaS 100%);a bone graft composition of the presently-disclosed subject matter thatincludes a commercial-grade calcium sulfate cement and that includes 10percent or 20 percent by volume of processed bone particles having ashape as shown in FIG. 1A (Tr 10% and Tr 20%, respectively); and a bonegraft composition that includes a calcium sulfate cement and thatincludes 10 percent or 40 percent by volume of non-specially shaped boneparticles (Alt 10% and Alt 40%, respectively);

FIG. 7 is a graph showing the results of a bending test performed with athree-point bending fixture as shown in FIG. 3 to analyze the bendingstrength of: a pure, commercial grade calcium sulfate cement (CaS 100%);and a bone graft composition of the presently-disclosed subject matterthat includes a commercial-grade calcium sulfate cement and thatincludes 10 percent by volume of processed bone particles having a shapeas shown in FIG. 1G (Elong 10%);

FIG. 8 is a schematic diagram of an experimental shear test fixture usedto apply a force (F) along a shear line and assess the shear strength ofa hardened bone graft composition (cylindrical specimen) made inaccordance with the presently-disclosed subject matter;

FIG. 9 is a graph showing the results of a shear test performed with ashear test fixture as shown in FIG. 8 to analyze the shear strength of:a pure calcium phosphate cement (CaP 100%) comprised of tetracalciumphosphate (TTCP), monocalcium phosphate (MCP), and calcium carbonate; abone graft composition of the presently-disclosed subject matter thatincludes a calcium phosphate cement comprised of TTCP, MCP and calciumcarbonate and that includes 10 percent or 20 percent by volume ofprocessed bone particles having a shape as shown in FIG. 1A (Tr 10% andTr 20%, respectively); and a bone graft composition that includes acalcium phosphate cement comprised of TTCP, MCP and calcium carbonateand that includes 10 percent or 40 percent by volume of non-speciallyshaped bone particles (Alt 10% and Alt 40%, respectively);

FIG. 10 is a graph showing the results of a shear test performed with ashear test fixture as shown in FIG. 8 to analyze the shear strength of:a pure, commercial-grade calcium phosphate cement (CaP* 100%) made fromalpha-tricalcium phosphate powder; a bone graft composition of thepresently-disclosed subject matter that includes a commercial-gradecalcium phosphate cement made from alpha-tricalcium phosphate powder andthat includes 10 percent or 20% by volume of processed bone particleshaving a shape as shown in FIG. 1A (Tr 10% or Tr 20%, respectively); anda bone graft composition that includes a commercial-grade calciumphosphate cement made from alpha-tricalcium phosphate powder and thatincludes 40 percent by volume of non-specially shaped bone particles(Alt 40%);

FIG. 11 is a graph showing the results of a shear test performed withshear test fixture as shown in FIG. 3 to analyze the shear strength of:a pure, commercial grade calcium sulfate cement (CaS 100%); and a bonegraft composition of the presently-disclosed subject matter thatincludes a commercial-grade calcium sulfate cement and that includes 10percent or 20 percent by volume of processed bone particles having ashape as shown in FIG. 1A (Tr 10% and TR 20%, respectively);

FIG. 12 is a schematic diagram of an experimental fixture used to applya force (F) and assess the diametral tensile strength of a hardened bonegraft composition (cylindrical specimen) made in accordance with thepresently-disclosed subject matter;

FIG. 13 is a graph showing the results of a diametral tensile testperformed with a fixture as shown in FIG. 12 to analyze the diametraltensile strength of: a pure, commercial grade calcium sulfate cement(CaS 100%); a bone graft composition of the presently-disclosed subjectmatter that includes a commercial-grade calcium sulfate cement and thatincludes 10 percent or 20 percent by volume of processed bone particleshaving a shape as shown in FIG. 1A (Tr 10% and TR 20%, respectively);and a bone graft composition that includes a calcium sulfate cement andthat includes 10 percent or 40 percent by volume of non-specially shapedbone particles (Alt 10% and Alt 40%, respectively);

FIG. 14 is a graph showing the bending strength of bone graftcompositions comprised of elongated cortical bone particles that weremixed with calcium phosphate cement, where the bone particles were addedto the calcium phosphate cement powder in approximate volume ratios of0.0% (X000), 1.25% (X125), 2.5% (X250), 3.75% (X375), and 5.0% (X500);

FIG. 15 is a graph showing the bending toughness of bone graftcompositions comprised of elongated cortical bone particles that weremixed with calcium phosphate cement, where the bone particles were addedto the calcium phosphate cement powder in approximate volume ratios of0.0% (X000), 1.25% (X125), 2.5% (X250), 3.75% (X375), and 5.0% (X500);

FIG. 16 is a graph showing the shear strength of bone graft compositionscomprised of elongated cortical bone particles that were mixed withcalcium phosphate cement, where the bone particles were added to thecalcium phosphate cement powder in approximate volume ratios of 0.0%(X000), 1.25% (X125), 2.5% (X250), 3.75% (X375), and 5.0% (X500);

FIG. 17 is a graph showing the shear toughness of bone graftcompositions comprised of elongated cortical bone particles that weremixed with calcium phosphate cement, where the bone particles were addedto the calcium phosphate cement powder in approximate volume ratios of0.0% (X000), 1.25% (X125), 2.5% (X250), 3.75% (X375), and 5.0% (X500);

FIG. 18 is a schematic diagram showing the ability of various shapedbone particles to interconnect with one another, where rectangular boneparticles and dumbbell-shaped bone particles are capable ofinterconnecting with one another along multiple surfaces, on the sidesand corners of the shaped particles, but where circular bone particlesare only capable of interconnecting with one another at single points ofcontact;

FIG. 19 is a schematic diagram showing the various models used todetermine the mechanical behavior effects of shaped cortical boneparticles in calcium phosphate cement, including a model that containedcalcium phosphate cement only (Model 1); a model that containedcylindrical cortical bone particles in calcium phosphate cement (Model2); a model that contained demineralized, cylindrical cortical boneparticles in calcium phosphate cement (Model 3); a model that containeddumbbell-shaped cortical bone particles in calcium phosphate cement(Model 4); and a model that contained demineralized, dumbbell-shapedcortical bone particles in calcium phosphate cement (Model 5);

FIG. 20 is an image showing the results of finite element analysis of amodel containing calcium phosphate cement only (Model 1), where thedarker areas represent regions of higher stress;

FIG. 21 is an image showing the results of finite element analysis of amodel containing cylindrical cortical bone particles in calciumphosphate cement (Model 2), where the darker areas represent regions ofhigher stress;

FIG. 22 is an image showing the results of finite element analysis of amodel containing demineralized, cylindrical cortical bone particles incalcium phosphate cement (Model 3), where the darker areas representregions of higher stress;

FIG. 23 is an image showing the results of finite element analysis of amodel containing dumbbell-shaped cortical bone particles in calciumphosphate cement (Model 4), where the darker areas represent regions ofhigher stress;

FIG. 24 is an image showing the results of finite element analysis of amodel containing demineralized, dumbbell-shaped cortical bone particlesin calcium phosphate cement (Model 5), where the darker areas representregions of higher stress;

FIG. 25 is a graph showing the amount of the area of calcium phosphatecement that was stressed in excess of its strength (>5 MPa) in thevarious models illustrated in FIG. 14;

FIGS. 26A-26D include light and fluorescent microscopy images ofcancellous bone defects in the lateral femoral condyles of rabbits thatwere filled with allograft cancellous bone obtained from other rabbitsor xenograft cancellous bone from young pigs, including images of theallograft-treated bone defects after 10 weeks with hematoxylin/eosinstaining (FIG. 26A) and with calcein labeling (FIG. 26B) to show theaddition of new bone, and images of xenograft-treated bone defects after10 weeks with hematoxylin/eosin staining (FIG. 26C) and with calceinlabeling to show the addition of new bone (FIG. 26D);

FIGS. 27A-27B include graphs illustrating the extent of cancellous boneincorporation (FIG. 27A) and the amount of inflammatory response (FIG.27B) observed in cancellous bone defects at various time points, wherethe bone defects were filled with either allograft cancellous boneobtained from other rabbits (Allograft) or xenograft cancellous bonefrom pigs (Xenograft);

FIGS. 28A-28B are images of computer-generated, three-dimensionalmicro-CT reconstructions showing the extent of remodeling and new boneformation in drill hole defects in the femoral condyles of rabbits,where the drill hole defects were filled with either hydroxyapatitecement only (FIG. 28A) or a mixture of hydroxyapatite cement andxenograft bone particles (FIG. 28B);

FIGS. 29A-29C include light microscopy images showing cellular activityand new bone formation in drill hole defects in the femoral condyles ofrabbits, where the drill hole defects were filled with eitherhydroxyapatite cement only (FIG. 29A) or a mixture of hydroxyapatitecement and xenograft bone particles (FIG. 29B and FIG. 29C);

FIGS. 30A-30B include graphs illustrating the extent of incorporationand new bone formation (FIG. 30A) and the amount of inflammation andcellular activity (FIG. 30B) observed in drill hole defects in thefemoral condyles of rabbits 10 weeks after the drill hole defects werefilled with either hydroxyapatite cement only (HAC) or a mixture ofhydroxyapatite cement and xenograft bone particles (XBC);

FIGS. 31A-31B and FIG. 31C include fluorescent microscopy images and agraph, respectively, showing new bone formation in calcein-labeled drillhole defects in the femoral condyles of rabbits 10 weeks after the drillhole defects were filled with either hydroxyapatite cement only (FIG.31A) or a mixture of hydroxyapatite cement and xenograft bone particles(FIG. 31B);

FIG. 32 is a graph showing the indentation strength of bone graftcompositions comprised of either hydroxyapatite cement only (HAC) or amixture of hydroxyapatite cement and xenograft bone particles (XBC) atthe time of inserting the composition or 10 weeks after inserting thecompositions in drill hole defects in the femoral condyles of rabbits;

FIG. 33 is a micro-computerized tomography (micro-CT) image of a drillhole defect in the lateral femoral condyle of a rabbit that was filledwith a bone graft composition of the presently-disclosed subject matterthat comprised calcium phosphate cement and dumbbell-shaped boneparticles;

FIG. 34 is a copy of the micro-CT image shown in FIG. 33, but with thecement-filled area of the bone defect highlighted and with the boneparticles within the cement further highlighted and shaded white;

FIG. 35 is a light microscopy image showing a cross-section of a drillhole defect in the lateral femoral condyle of a rabbit that was filledwith a bone graft composition comprising calcium phosphate cement andallograft, dumbbell-shaped bone particles;

FIG. 36 is a light microscopy image showing a portion of the microscopyimage shown in FIG. 35 at a higher magnification (100×), and furthershowing cellular activity from the defect boundary into the boneparticle via the demineralized layer and a boundary between thedemineralized bone layer and the mineralized bone;

FIG. 37 is another light microscopy image showing a portion of themicroscopy image shown in FIG. 35 at a higher magnification (100×), andfurther showing cellular activity and incorporation of an allograftparticle from the defect boundary via the demineralized layer and aboundary between the demineralized bone layer and the mineralized bone;

FIG. 38 is also a light microscopy image showing a portion of themicroscopy image shown in FIG. 35 at a higher magnification (100×), andfurther showing cellular infiltration and incorporation of theallograft, dumbbell-shaped bone particles;

FIGS. 39A-39C are images showing a three-dimensional micro-CTreconstruction of the distal femur region of a rabbit, where a drillhole defect in that region was filled with a bone graft composition ofthe presently-disclosed subject matter comprising calcium phosphatecement and dumbbell-shaped bone particles, including an image of theentire distal femur region (FIG. 39A), an image showing a transversetrim of the reconstruction through the middle of the defect (FIG. 39B),and an image where the reconstruction has been trimmed from the top andfront (FIG. 39C);

FIGS. 40A-40B are images showing an approximately 2 mm thick slabmicro-CT reconstruction of the distal femur region of a rabbit, where adrill hole defect in that region was filled with a bone graftcomposition of the presently-disclosed subject matter comprising calciumphosphate cement and dumbbell-shaped bone particles, where FIG. 40Bisolates the lower-density demineralized layer covering eachspecially-shaped bone particle;

FIGS. 41A-41B are images showing an approximately 5 mm thick slabmicro-CT reconstruction of the distal femur region of a rabbit, where adrill hole defect in that region was filled with a bone graftcomposition of the presently-disclosed subject matter comprising calciumphosphate cement and dumbbell-shaped bone particles, where FIG. 41Bisolates the lower-density demineralized layer covering eachspecially-shaped bone particle;

FIGS. 42A-42B are graphs showing the instantaneous (FIG. 42A) andcumulative (FIG. 42B) lysozyme release from bone graft compositions ofthe presently-disclosed subject matter, where the lysozyme waspreadsorbed onto the bone particles prior to adding the bone particlesto a calcium phosphate cement;

FIGS. 43A-43B are graphs showing the instantaneous (FIG. 43A) andcumulative (FIG. 43B) vancomycin release from bone graft compositions ofthe presently-disclosed subject matter, where the vancomycin wasdry-mixed with the calcium phosphate cement and bone particles prior tothe addition of an aqueous vehicle to set the mixture;

FIGS. 44A-44DD are images of serial sections of a bone graft compositionof the presently-disclosed subject matter showing the distribution andinterconnectedness of the plurality of processed bone particles that areincluded in the composition, where each of the processed bone particleshas a shape as shown in FIG. 1A;

FIGS. 45A-45E are schematic diagrams showing: a screw placed incancellous bone (FIG. 45A); a screw placed in cancellous bone, where theplacement of the screw is augmented with cement (FIG. 45B); a failure ofa screw placed in a cancellous bone, where the placement of the screwwas augmented with cement, and where the failure occurs at the interfaceof the cancellous bone and the cement (FIG. 45C); a failure of a screwplaced in a cancellous bone, where the placement of the screw wasaugmented with cement, and where the failure occurs via shear force atthe interface of the screw and the cement (FIG. 45D); and a screw placedin cancellous bone, where the placement of the screw is augmented withcement and processed bone particles having a shape as shown in FIG. 1A,and where the processed bone particles bridge across and strengthen boththe screw-cement and cement-bone interfaces (FIG. 45E); and

FIG. 46 is a graph showing the results of a dynamic bending toughnesstest performed to analyze the bending toughness of: a pure calciumphosphate cement (CaP 100%) comprised of tetracalcium phosphate (TTCP),monocalcium phosphate (MCP), and calcium carbonate; and a bone graftcomposition of the presently-disclosed subject matter that includes acalcium phosphate cement comprised of TTCP, MCP and calcium carbonateand that includes 10 percent by volume of processed bone particleshaving a shape as shown in FIG. 1A (Tr 10).

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding, and no unnecessary limitations are to be understoodtherefrom.

While the following terms are believed to be well understood by one ofordinary skill in the art, definitions are set forth to facilitateexplanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.Although many methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a bone particle” includes aplurality of such particles, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations in some embodiments of ±20%, in someembodiments of ±10%, in some embodiments of ±5%, in some embodiments of±1%, in some embodiments of ±0.5%, and in some embodiments of ±0.1% fromthe specified amount, as such variations are appropriate to perform thedisclosed method.

Bone grafting is a surgical procedure that replaces missing bone withautograft, allograft, or xenograft bone materials, or a suitable bonegraft composition. Bone grafting is possible because bone, unlike manyother tissues, has the ability to regenerate completely if it isprovided with the proper conditions and space in which to grow. For anybone graft composition to be effective and allow natural bone to fullyoccupy the space of a previous defect, however, several importantqualities must be taken into consideration including: strength ormechanical stability (i.e., the ability to maintain physicalrelationships between bone surfaces into which the material is placed);osteoconductivity (i.e., the capability to function as a scaffold ontowhich new bone can form); and, in some instances, osteoinductivity(i.e., the property of stimulating migration and proliferation of bonecells in the subject to grow and become active at the graft site). Assuch, a synthetic bone graft composition should provide immediatemechanical stability and resorb quickly, but yet should also be able toeffectively promote new bone formation. To that end, thepresently-disclosed subject matter includes novel bone graftcompositions that are comprised of bone particles of varying shapes,sizes, and quantities such that the bending strength and toughness,shear strength and toughness, tensile strength and toughness, andincorporation and remodeling rates of the compositions are optimized.

In some embodiments of the presently-disclosed subject matter, a bonegraft composition is provided that includes materials added to abiologically-resorbable cement, which allow the cement structure thatforms, after it sets in the body, to more rapidly incorporate andremodel. In some embodiments, the materials that are added to thecompositions include processed bone particles, which allow thecompositions to remodel faster when placed in a subject, but also allowfor infiltration of the cement structure by cells, blood, and other suchbodily fluids and structures.

In some embodiments of the presently-disclosed subject matter, bonegraft compositions are provided that include specially-shaped, processedbone particles. In some embodiments, a bone graft composition isprovided that comprises a biologically-resorbable cement and a pluralityof processed bone particles. As described in further detail below, inthese compositions, each of the processed bone particles has a shapesuch that is configured to interconnect and interlock with adjacent boneparticles, the surrounding cement, or both when it is included in a bonegraft composition of the presently-disclosed subject matter.

The term “biologically-resorbable cement” is used herein to refer to anybiological cement, such as a bone substitute cement, that is capable ofbeing broken down and assimilated by the body of a subject, and that issubstantially non-toxic in the in vivo environment of its intended usesuch that it is not substantially rejected by the subject'sphysiological system (i.e., is non-antigenic or biocompatible). This canbe gauged by the composition's toxicity, infectivity, pyrogenicity,irritation potential, reactivity, hemolytic activity, carcinogenicityand/or immunogenicity. A biologically-resorbable cement, when introducedinto a bone of a majority of subjects, will not cause an undesirablyadverse, long-lived or escalating biological reaction or response, andis distinguished from a mild, transient inflammation which typicallyaccompanies surgery or implantation of foreign objects into a livingorganism.

As would be recognized by those skilled in the art, a “cement” is aproduct that is produced as a result of the setting of a paste that isformed by mixing a powdered component with water or another aqueousvehicle. A number of biologically-resorbable cements can be formed bymixing a powder component with water or another aqueous vehicle and thenused in accordance with the presently-disclosed bone graft compositions,including, but not limited to, ceramics-based cements, calcium-basedcements, magnesium ammonium-based cements, and the like. In someembodiments of the presently-disclosed compositions, thebiologically-resorbable cement is a calcium-based cement, such as acalcium sulfate cement or a calcium phosphate cement, where the powderedcomponent is comprised of a calcium-based compound. In some embodiments,the calcium-based cement is a calcium phosphate cement. In otherembodiments, the calcium-based cement is a calcium sulfate cement.

The phrase “calcium phosphate cement” is used herein to refer to acement where the powdered component of the cement is comprised of acalcium phosphate compound or a mixture of calcium and/or phosphatecompounds. Exemplary calcium phosphate compounds or mixtures of calciumcompounds and/or phosphate compounds that can be mixed with water oranother aqueous vehicle and used in accordance with thepresently-disclosed subject matter include, but are not limited to:tricalcium phosphate (Ca₃(PO₄)₂; TCP), including alpha-TCP, beta-TCP,and biphasic calcium phosphate containing alpha- and beta-TCP; amorphouscalcium phosphate (ACP); monocalcium phosphate (Ca(H₂PO₄)₂; MCP) andmonocalcium phosphate monohydrate (Ca(H₂PO₄)₂.H₂O; MCPM); dicalciumphosphate (CaHPO₄; DCP), dicalcium phosphate anhydrous (CaHPO₄; DCPA)and dicalcium phosphate dihydrate (CaH₅PO₆.2H₂O; DCPD); tetracalciumphosphate ((Ca₄PO₄)₂O; TTCP); octacalcium phosphate(Ca₈(PO₄)₄HPO₄)₂.5H₂O; OCP); calcium hydroxyapatite (Ca_(m)(PO₄)₆(OH)₂;CHA); calcium oxyapatite (Ca_(m)(PO₄)₆O; COXA); calcium carbonateapatite (Ca₁₀(PO₄)₆CO₃; CCA); and calcium carbonate hydroxyapatites(e.g., Ca₁₀(PO₄)₅(OH)(CO₃)₂ and C₁₀(PO₄)₄(OH)₂(CO₃)₃; CCHA). Additionalcalcium phosphates useful herein also include calcium-deficient calciumphosphates in which the molar or mass ratio of Ca:P is reduced by about20% or less, about 15% or less, or about 10% or less, relative to thecorresponding calcium non-deficient species, examples of which includecalcium-deficient hydroxyapatites, e.g.,Ca_(10-x)(HPO₄)_(x)(PO₄)_(6-x)(OH)_(2-x) (O≤X≤1) (CDHA);calcium-deficient carbonate hydroxyapatites (CDCHA); calcium-deficientcarbonate apatites (CDCA); and other calcium phosphate compounds andsalts known to be useful in the field of bone graft materials, e.g.,calcium polyphosphates; and calcium-, phosphate-, and/or hydroxyl“replaced” calcium phosphates. In some embodiments, thecalcium-phosphate cement is a hydroxyapatite cement. For furtherexplanation and guidance regarding calcium phosphate cements, see, e.g.,Ambard, et al. Journal of Prosthodontics. 15(5): 321-326 (2006).

The phrase “calcium sulfate cement” is used herein to refer to a cementwhere the powdered component of the cement is comprised of a calciumsulfate compound or a mixture of calcium and/or sulfate compounds.Exemplary calcium sulfate compounds or mixtures of calcium compoundsand/or sulfate compounds that can be mixed with water or another aqueousvehicle and used in accordance with the presently-disclosed subjectmatter include, but are not limited to: calcium sulfate (CaSO₄); calciumsulfate dihydrate (2CaSO₄.2H₂O); and calcium sulfate hemihydrate(CaSO₄.½H₂O). For further explanation and guidance regarding calciumsulfate cements, see, e.g., Bohner, European Cells & Materials, Vol. 20,2010, pages 1-12.

Turning now to the processed bone particles that are included in thepresently-disclosed bone graft compositions, the phrase “processed boneparticles” is used herein to refer to pieces of bone that are derivedfrom an intact bone, or part of an intact bone, and have been modifiedto produce pieces of bone with a desired level of mineralization, adesired size, and/or a desired shape, such that the pieces of bone canbe combined with a suitable cement and applied to the site of a bonedefect, as described in detail below. In some embodiments, the processedbone particles are of a size and shape that allows a prescribed mixtureof cement (e.g., calcium phosphate cement) and processed bone particlesto flow in a paste-like consistency, similar to the handlingcharacteristics of processed cement. In some embodiments, the processedbone particles are from an autograft bone source, an allograft bonesource, a xenograft bone source, or combinations thereof.

To produce an exemplary bone graft composition of thepresently-disclosed subject matter, a specially-shaped bone particle isfirst obtained by refining an intact whole bone into a number ofdiscrete particles. For example, to obtain a cortical bone particlehaving an interconnecting shape, as also described in further detailbelow, the soft tissue is first removed from the diaphysis of an intactbone, and the distal and proximal ends of the bone are removed. The bonemarrow and the soft tissue inside the bone's shaft are then removed, andthe inside of the bone is rinsed out, subsequent to removing anyremaining cancellous bone from the inside of the diaphysis. The shaft ofbone is then cut into thinned pieces of cortical bone, and is thentypically either inserted into a punch that cuts the bone pieces into adesired interconnecting shape with a desired thickness or, in someembodiments, is mounted in a lathe to produce bone particles having adesired interconnecting shape. Of course, other techniques known tothose of ordinary skill in the art including laser cutting techniquesand the like can also be used to produce bone particles having a desiredinterconnecting shape and can be used without departing from the spiritand scope of the subject matter described herein.

The cement mixtures of the bone graft compositions are generally formedby mixing the powdered component of the cement with water or anotheraqueous vehicle. In this regard, once the specially-shaped boneparticles are formed, the bone particles are then mixed with the cementat a desired concentration, as described further below. The term“aqueous vehicle” is used herein to refer to any fluid, such as water,that can be mixed with a powdered component of a cement to form asuitable paste of a biologically-resorbable cement. In this regard, theaqueous vehicle must also be substantially non-toxic in the in vivoenvironment of its intended use such that it is not substantiallyrejected by the subject's physiological system. In addition to water,such aqueous vehicles can include, but are not limited to, bufferedsaline solutions, sodium phosphate monobasic monohydrate (NaH₂PO₄.H₂O)solutions, sodium phosphate dibasic (Na₂HPO₄) solutions, glycerolsolutions, and the like.

Typically, the amount of water or other aqueous vehicle that is mixedwith the powdered component of the cement and the specially-shapedprocessed bone particles of the presently-disclosed subject matter is atleast enough to generate the standard chemical reaction for cementsetting to occur. When the bone particles are mixed with the cement, theaqueous vehicles temporarily hydrate any exposed collagen in theprocessed bone particles to allow the bone graft compositions toinitially have flow and adherence properties of a standard processedcement. As the water is consumed, the collagen then binds with itssurroundings and, at this point, any excess water, or other aqueousvehicle, beyond what is needed for the cement reaction to occur, can betaken up by the porosity of the bone particles or the exposed collagen.In some embodiments, the amount of water absorbed or adsorbed by theparticles is about 30 percent to about 50 percent of the weight of thedry bone particles, such that, in certain embodiments, the amount ofwater or other aqueous vehicle absorbed or adsorbed by the boneparticles comprises about 10 percent to about 20 percent of the volumeof aqueous vehicle necessary for the setting reaction to occur.

As noted above, the bone particles of the presently-disclosed bone graftcompositions have a shape that is configured to interconnect withadjacent bone particles when a plurality of the bone particles areincluded in a bone graft composition of the presently-disclosed subjectmatter. The terms “interconnect” or “interconnecting” as used herein inreference to the processed bone particles refer to bone particles havingshapes that include intersecting surfaces or other structural featuresthat allow the bone particles to interlock and/or more readily interactwith one another, as opposed to simple cylindrical or spherical boneparticles that would be unable to interlock with one another or would beless efficient at creating interconnected pathways by virtue of theassociation of one bone particle with one or more additional, adjacentbone particles.

For example, in some embodiments and as shown in FIGS. 1A-1C and 1E, thebone particles 10, 110, 210, 410 are dumbbell-shaped, such that when thedumbbell-shaped bone particles 10, 110, 210, 410 are included in a bonegraft composition of the presently-disclosed subject matter, theenlarged ends 14 a, 14 b, 114 a, 114 b, 214 a, 214 b, 414 a, 414 b ofthe dumbbell-shaped bone particles 10, 110, 210, 410 overlap and allowcontact and engagement of the dumbbell-shaped bone particles alongmultiple surfaces (see, e.g., FIGS. 2 and 18). In some embodiments ofthe dumbbell-shaped bone particles, and as also shown in FIGS. 1A-1C and1E, each bone particle 10, 110, 210, 410 includes two enlarged endportions 14 a, 14 b, 114 a, 114 b, 214 a, 214 b, 414 a, 414 b thatextend laterally away from a longitudinal axis of the center portion 12,112, 212, 412 of each bone particle. In some embodiments of thedumbbell-shaped bone particles, and as shown in FIG. 1B, adumbbell-shaped bone particle 110 is provided that includes a centerportion 112 with a circular cross-section and two disc-shaped endportions 114 a, 114 b that extend laterally away from (e.g., areoriented in a direction perpendicular to) the longitudinal axis of thecenter portion 112. In further embodiments, and as shown in FIG. 1C, adumbbell-shaped bone particle 210 is provided that includes a centerportion 212 with a generally elliptical cross-section and substantiallysquare ends 214 a, 214 b that extend laterally away from thelongitudinal axis of the center portion 212. In yet other embodiments,and as show in FIG. 1E, a dumbbell-shaped bone particle 410 is providedthat includes a substantially-flat top surface 418 and asubstantially-flat bottom surface 416, and further includes a centerportion 412 with a generally square cross-section, and rectangular endportions 414 a, 414 b that laterally extend away from and are orientedin a direction perpendicular to the longitudinal axis of the centerportion 412. Of course, to the extent it may be desired, bone particlesof various other interconnecting shapes that would be capable ofconnecting with one another on multiple surfaces, such as “S-shaped” or“T-shaped” or “C-shaped” bone particles, can also be produced and usedin a bone graft composition of the presently-disclosed subject matterwithout departing from the spirit and scope of the subject matterdescribed herein.

Furthermore, in certain embodiments, a number of interconnecting shapeshaving increased lengths can be provided that are capable ofinterconnecting with one another on multiple surfaces. For example, andas shown in FIG. 1D, in some embodiments, an elongated bone particle 310is provided that includes a plurality of rectangular portions 314 and aplurality of center portions 312 aligned along a common longitudinalaxis. In the bone particle 310, each of the rectangular portions 314 areoriented in a direction perpendicular to the common longitudinal axis ofeach center portion 312 and each of the center portions 312 areinterposed between the respective rectangular portions 314. As anotherexample of an elongated bone particle made in accordance with thepresently-disclosed subject matter, and as shown in FIG. 1F, anelongated bone particle 510 is provided that includes a plurality ofenlarged, spherical portions 514 and a plurality of center portions 512aligned along a common longitudinal axis, where each of the enlargedportions 514 extend laterally away from the common longitudinal axis ofeach center portion 512, and where each of the center portions 512 areinterposed between respective enlarged portions 514.

In the embodiment shown in FIG. 1F, the bone particle 510 includes threeenlarged portions 514. However, it is further contemplated that anynumber of enlarged portions can be included in a specially-shaped boneparticle to produce bone particles of varying lengths without departingfrom the spirit and scope of the subject matter described herein. Forinstance, and as shown in FIG. 1G, an elongated bone particle 610 isprovided that resembles a number of dumbbell-shaped bone particlesplaced end-to-end and includes five enlarged, spherical portions 614 anda plurality of center portions 612 aligned along a common longitudinalaxis, where each of the enlarged portions 614 also extend laterally awayfrom the common longitudinal axis of each center portion 612, and whereeach of the center portions 612 are also interposed between respectiveenlarged portions 614.

In some embodiments of the presently-disclosed subject matter, theinterconnectedness of the bone particles also increases the compressive,bending, tensile, and shear strength of the bone graft compositions(i.e., the combination of particles and cement) by providing directloading pathways through contacting other bone particles, which arestronger than the cement matrix. In this regard, in some embodiments,the interconnectedness of the bone particles is increased by eachparticle having larger dimensions at its ends compared to its center.For example, the inclusion of bone particles having a dumbbell shape, asdescribed above, or a shape in the form of a capital “I” will have anincreased connection to adjacent bone particles when compared to boneparticles having a shape in the form of a capital “O,” assuming bothshapes have similar length and width.

In some embodiments, the interconnecting of the bone particles allowsthe particles to increase their resistance to relative elongationdisplacement, including when they are embedded in a hardened cement. Insome embodiments, the bone particles are further configured tointerdigitate with the biologically-resorbable cement such that thestrength and mechanical benefits of the presently-disclosed bone graftcompositions are further increased. By including interconnecting boneparticles in a bone graft composition, the bone particles are able to,in some embodiments, interlock and strengthen the bone graftcompositions by the “keystoning” of the cement matrix, a term which isused herein to describe the conversion of tension in the shapedparticles to compression in the cement matrix because of the directinteraction between the particle surfaces and the cement contactingsurfaces. For an illustration and further guidance regarding keystoningof a cement matrix, see, e.g., FIGS. 18 and 19.

Additionally, the interconnecting of the bone particles also contributeto the enhanced incorporation, remodeling, and resorption of the bonegraft compositions when the compositions are placed in a bone defect invivo by extending three-dimensionally throughout the bone graftcomposition and bone defect site, and increasing the likelihood that thebone particles communicate not only with one another, but with thefluids and cells outside the cement surface. In other words, byincluding bone particles having an interconnecting shape in a bone graftcomposition, portions of the bone particles are capable of extendingthroughout the composition and into and through the outer surface of thecement structure that is formed when the bone graft composition setsinto a solid structure in vivo, which, in turn, allows the compositionto be accessible to cells and fluids (e.g., blood supply) from thesubject and, ultimately, allows the bone graft composition to beincorporated into a subject.

For a bone graft composition to achieve the objective of becomingcompletely incorporated into a subject once it is placed in a bonedefect, the bone graft composition must generally be rapidly remodeledand replaced with living bone in as short of time as possible, orremodeled such that a new trabecular architecture is restored within thegeometry formed by the hardened cement having an interconnected networkof included bone graft shapes. As such, it is thought that, not onlymust the bone graft composition be completely incorporated into a host,but the bone particles included in the composition must achieve a“cross-sample bioconnectivity,” where the bone particles extend throughthe composition, once it is placed at the site of a bone defect, andcommunicate with each other and the outer surface of the bone graftcomposition to allow access to the grafted region by various cells andfluid from the subject. In this regard, it is also generally thoughtthat as much bone material (i.e., bone particles) should be incorporatedinto a cement-based bone graft composition as possible and that the bonematerial should be readily accessible to the cells of a subject and theblood supply of a subject. However, the inclusion of an excessive amountof bone material in a cement-based bone graft composition frequentlyleads to a bone graft composition that does not exhibit the requiredmechanical stability and that does not allow the cement to behave like acement in terms of the handling, flowability, and settingcharacteristics. Conversely, the inclusion of too little an amount ofbone material in a cement-based bone graft composition often leads to abone graft composition that is not sufficiently incorporated into asubject.

It has been experimentally observed, however, that the bone graftcompositions of the presently-disclosed subject matter, which make useof bone particles having interconnecting shapes, are capable ofoptimizing the cross-sample bioconnectivity of the bone graftcomposition, while still preserving the mechanical stability of the bonegraft composition itself (see, e.g., FIGS. 44A-44DD, showing images ofserial sections of a bone graft composition, where, from one image tothe next, the bone particles and, more specifically, the outerdemineralized layers (shown in white) of the bone particles can be seento connect to one another and to the outer surface of the cement (shownin black)). In particular, it has been determined that theinterconnecting bone particles allow for an increased amount of cementto be present in the compositions, as compared to cement-basedcompositions that include only simple-shaped bone particles (e.g.,cylindrical or spherical bone particles), such that thepresently-disclosed bone graft composition is able to behave like acement in terms of its handling, flowability, and settingcharacteristics. However, it has also been determined that by includingthe bone particles having an interconnecting shape in thepresently-disclosed bone graft compositions, the compositions areallowed to behave as a cement while the bone particles provide aninterlocking mechanical construct that augments the mechanicalproperties of the final cement volume once it has been administered toand has set up at the site of a bone defect. In some embodiments, thecross-sections of the processed bone particles described above may besubstantially round, elliptical, square, rectangular, triangular, orhave another prismatic shape such that the bone particles are able tofurther strengthen the cement in shear, tension, and bending loadingmodes as compared to the cement in its uncomposited form or as comparedto cement that includes randomly-shaped and/or randomly-orientedparticles. In some embodiments of the presently-disclosed bone graftcompositions, the interconnecting bone particles are combined with thebiologically-resorbable cement at a concentration of about 1 percent,about 2 percent, about 3 percent, about 4 percent, about 5 percent,about 6 percent, about 7 percent, about 8 percent, about 9 percent,about 10 percent, about 11 percent, about 12 percent, about 13 percent,about 14 percent, about 15 percent, about 20 percent, about 25 percent,about 30 percent, about 35 percent, about 40 percent, about 45 percent,about 50 percent by volume of the bone graft composition. In someembodiments, the interconnecting bone particles are combined with thebiologically-resorbable cement at a concentration of about 1 percent toabout 50 percent by volume of the bone graft composition. In someembodiments, the interconnecting bone particles are combined with thebiologically-resorbable cement at a concentration of about 1 percent toabout 15 percent by volume of the bone graft composition.

In some embodiments, the interconnecting bone particles of thepresently-disclosed subject matter allow for an increased incorporationand remodeling of the bone graft composition, as compared tocement-based compositions that include only simple-shaped bone particles(e.g., cylindrical or spherical bone particles), by providing boneparticles having an increased surface area. As will be recognized bythose skilled in the art, the formation and infiltration of new bone atthe site of a bone graft is a surface-driven phenomenon with the surfacetopology of the graft being capable of encouraging or hindering new bonefrom populating the graft site. By providing bone particles having aninterconnecting shape, the interconnecting bone particles generallyprovide a greater surface area and off-axis span, as compared to boneparticles having a simple shape such as a sphere or cylinder, whichincreases the likelihood of multiple bone particles touching andinterconnecting and interlocking with each other in the composition, andalso increases the osteoconductivity and osteoinductivity of thecompositions. In this regard, the inclusion of the interconnecting boneparticles in a bone graft composition results in a network ofinterconnected pathways or channels that allow for cells and fluids fromthe subject to infiltrate the bone graft composition and the bone graftitself, leading to the incorporation of the bone graft composition andits replacement with living bone from the subject. In other words, insome embodiments, the interconnecting bone particles can convey the hostfluids and cells into the interior of the bone graft composition inorder to allow resorption and new bone formation throughout thematerial, rather than only on its most exterior surface.

In some embodiments, the infiltration and activity of cells and fluidsfrom the subject depends, at least in part, on the type of bone that isused to fabricate the bone particles of the presently-disclosed subjectmatter. In some embodiments, the processed bone particles comprisecancellous bone particles that are capable of creating a pathway throughthe bone graft composition and the bone graft without the need to modifytheir surface prior to including the cancellous bone particles in thecomposition. As would be recognized by those skilled in the art,cancellous or spongy bone is comprised of collagenous trabeculae and istypically less dense than cortical bone. As such, when cancellous boneis used to fabricate a bone particle having a interconnecting shape, thetrabeculae provide tunnel-like spaces in the bone particles that can beused by the cells and fluids of the subject to infiltrate the bone graftand cause the incorporation and resorption of the bone graftcomposition.

In other embodiments of the presently-disclosed bone graft compositions,the processed bone particles are comprised of cortical bone. In theseembodiments, the outer surface of cortical bone is typically firstdemineralized to provide a means to facilitate the movement of cells andfluids to the interior of the bone graft. The term “demineralized” isused herein to refer to the process by which bone mineral or theinorganic portion of the bone is removed to thereby expose the collagenportion of the bone. In this regard, in some embodiments, to prepare abone particle of the presently-disclosed subject matter (e.g., acortical bone particle), a demineralization process can be used suchthat the outer surface of the bone is transformed into an exposedcollagen layer that is then capable of stimulating and facilitating theinfiltration and activity of cells and fluid from the subject into thebone graft. In some embodiments, the processed bone particles are about5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, to about 90% demineralized. Insome embodiments, by demineralizing the bone particles, the speed withwhich the bone graft composition is incorporated, remodeled, or resorbedinto the subject and replaced by living bone is increased, while thebone graft composition maintains and improves the strength of thesubject's bone and the graft itself. In some embodiments, if theprocessed bone particles are not from an autograft source, thedemineralization of the processed bone particles can increase the rateat which the bone graft composition is incorporated into the subject andreplaced with living bone from the subject.

As noted above, in some embodiments that make use of bone particleshaving a demineralized layer, the interconnectedness of the boneparticles of the presently-disclosed subject matter further increasesthe interconnectedness of the osteoinductive demineralized layerscovering each particle. In this regard, in certain embodiments, theinterconnected network of demineralized bone matrix (DBM) is oriented toresemble a restored trabecular architecture in the incorporating cementmaterial mass, and the specific thickness of the demineralized layer canaid in the osteoconductivity of the end product. In some embodiments,the formation of a demineralized layer, while providing a pathway forthe stimulation of activity and the infiltration of cells and fluid intothe grafted region, as well as rapid resorption of the bone graftcomposition, also allows for the addition of an osteoinductive material,an osteogenic material, or both to the surface of the bone particles tothereby further enhance the incorporation of the bone graft compositioninto the subject and its replacement with living bone from the subject.

The term “osteoinductive material” is used herein to refer to anymaterial that stimulates the migration or differentiation of bone cellsto grow and become active at a graft site, while the term “osteogenicmaterial” is used herein to refer to any material that is capable ofdirectly or indirectly contributing to the action of osteoblasts orother cells capable of contributing to new bone growth. In someembodiments, the osteoinductive material that is added to thedemineralized bone particles is selected from protein growth factorssuch as bone morphogenetic proteins (BMPs) and other proteins from thetransforming growth factor-beta superfamily. In some embodiments, theosteogenic materials that can be added to the demineralized boneparticles include host cells (e.g., osteoblasts, etc.) or stem cells orprogenitor cells.

To add an osteoinductive and/or an osteogenic agent to the exposedcollagen surface of a demineralized bone particle, the processed boneparticles can be soaked in a solution containing the osteoinductiveagent, the osteogenic agent, or both, prior to mixing the demineralizedbone particles with the biologically-resorbable cement, such that theosteoinductive and/or osteogenic agent simply incorporates into andadheres to the collagen surface. Of course, a number of other methodsfor linking such an agent to a protein such as collagen are known tothose of ordinary skill in the art and can be used without departingfrom the spirit and scope of the subject matter described herein.

In some embodiments, stem cells can further be added to the boneparticles to enhance the incorporation of the bone graft compositioninto the subject and its replacement with living bone from the subject.As used herein, the term “stem cells” refers broadly to traditional stemcells, progenitor cells, preprogenitor cells, precursor cells, bloodcells, platelets, reserve cells, and the like. Exemplary stem cellsinclude, but are not limited to, embryonic stem cells, adult stem cells,pluripotent stem cells, neural stem cells, muscle stem cells, muscleprecursor stem cells, endothelial progenitor cells, bone marrow stemcells, chondrogenic stem cells, lymphoid stem cells, mesenchymal stemcells, hematopoietic stem cells, and the like. Descriptions of stemcells, including methods for isolating and culturing them, may be foundin, among other places, Embryonic Stem Cells, Methods and Protocols,Turksen, ed., Humana Press, 2002; Weisman et al., Annu Rev. Cell. Dev.Biol. 17:387-403; Pittinger et al., Science, 284:143-47, 1999; AnimalCell Culture, Masters, ed., Oxford University Press, 2000; Jackson etal., PNAS 96(25):14482-86, 1999; Zuk et al., Tissue Engineering,7:211-228, 2001; and U.S. Pat. Nos. 5,559,022, 5,672,346 and 5,827,735.

In addition to adding various osteoinductive or osteogenic agents, suchas stem cells, to the bone particles of the presently-disclosed subjectmatter, it is further contemplated that a number of additionaltherapeutic agents can also be added directly to thebiologically-resorbable cement prior to mixing it with the processedbone particles. Without wishing to be bound by any particular theory, itis contemplated that the accelerated remodeling and incorporation of thecement due to the presence of the processed bone particles canfacilitate a more rapid and more complete release of a therapeutic agentinto the subject at the implantation site.

Further therapeutic agents that can be added to thebiologically-resorbable cement prior to or after mixing it with theprocessed bone particles include, but are not limited to: collagen andinsoluble collagen derivatives; hydroxyapatite; bisphosphonates and/orother anti-osteoporosis drugs; antiviricides, such as those effectiveagainst HIV and hepatitis; amino acids, peptides, vitamins, and/orco-factors for protein synthesis; hormones; endocrine tissue or tissuefragments; synthesizers; enzymes, such as collagenase, peptidases,oxidases; polymer cell scaffolds with parenchymal cells; angiogenicdrugs and polymeric carriers containing such drugs; collagen lattices;biocompatible surface active agents; antigenic agents; cytoskeletalagents; cartilage fragments; living cells, such as chondrocytes, bonemarrow cells, mesenchymal stem cells; natural extracts; tissuetransplants; bioadhesives; transforming growth factor (TGF-beta);insulin-like growth factor (IGF-1); parathyroid hormone; growthhormones, such as somatotropin; bone digesters; antitumor agents;fibronectin; cellular attractants and attachment agents;immuno-suppressants; and, permeation enhancers, e.g. fatty acid esterssuch as laureate, myristate and stearate monoesters of polyethyleneglycol, enamine derivatives, and alpha-keto aldehydes.

In some embodiments, an antibiotic is added to thebiologically-resorbable cement (e.g., the biologically-resorbable cementpowder) prior to mixing it with the processed bone particles of thepresently-disclosed subject matter. Various antibiotics can be employedin accordance with the presently-disclosed subject matter including, butare not limited to: aminoglycosides, such as amikacin, gentamycin,kanamycin, neomycin, netilmicin, paromomycin, streptomycin, ortobramycin; carbapenems, such as ertapenem, imipenem, meropenem;chloramphenicol; fluoroquinolones, such as ciprofloxacin, gatifloxacin,gemifloxacin, grepafloxacin, levofloxacin, lomefloxacin, moxifloxacin,norfloxacin, ofloxacin, sparfloxacin, or trovafloxacin; glycopeptides,such as vancomycin; lincosamides, such as clindamycin;macrolides/ketolides, such as azithromycin, clarithromycin,dirithromycin, erythromycin, or telithromycin; cephalosporins, such ascefadroxil, cefazolin, cephalexin, cephalothin, cephapirin, cephradine,cefaclor, cefamandole, cefonicid, cefotetan, cefoxitin, cefprozil,cefuroxime, loracarbef, cefdinir, cefditoren, cefixime, cefoperazone,cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime,ceftriaxone, or cefepime; monobactams, such as aztreonam;nitroimidazoles, such as metronidazole; oxazolidinones, such aslinezolid; penicillins, such as amoxicillin, amoxicillin/clavulanate,ampicillin, ampicillin/sulbactam, bacampicillin, carbenicillin,cloxacillin, dicloxacillin, methicillin, mezlocillin, nafcillin,oxacillin, penicillin G, penicillin V, piperacillin,piperacillin/tazobactam, ticarcillin, or ticarcillin/clavulanate;streptogramins, such as quinupristin/dalfopristin; sulfonamide/folateantagonists, such as sulfamethoxazole/trimethoprim; tetracyclines, suchas demeclocycline, doxycycline, minocycline, or tetracycline; azoleantifungals, such as clotrimazole, fluconazole, itraconazole,ketoconazole, miconazole, or voriconazole; polyene antifungals, such asamphotericin B or nystatin; echinocandin antifungals, such ascaspofungin or micafungin, or other antifungals, such as ciclopirox,flucytosine, griseofulvin, or terbinafine. In some embodiments, theantibiotic that is included in a bone graft composition of thepresently-disclosed subject matter is vancomycin. For furtherexplanation and guidance regarding the use of cements, such as calciumphosphate cements, as drug delivery systems, see, e.g., Verron, et al.Drug Discovery Today. 15(13/14): 547-552 (2010).

As one particular example of adding a therapeutic, osteoinductivematerial to a bone graft composition of the presently-disclosed subjectmatter, bone morphogenetic protein 2 (BMP-2) can be mixed with thebiologically-resorbable cement at a ratio of 0.17 mg BMP-2 for every 1 gof cement. As another particular example of the addition of atherapeutic agent to a bone graft composition of the presently-disclosedsubject matter, 30 mg of a desired antibiotic, such as vancomycin, canbe added per gram of cement and can be mixed in powder form directlywith the resorbable cement powder prior to implantation. As yet anotherparticular example of adding a therapeutic osteogenic agent to a bonegraft composition of the presently-disclosed, a bisphosphonate can beadded for local bone quality improvement and preservation in a mannerthat has distinct advantages over other approaches. In this regard, insome embodiments, the bisphosphonate can be mixed directly with thepowder components of the cement so that it is evenly distributed withthe final set cement mass, and causing the release of the bisphosphonateto be dependent on the rate of cement resorption by osteoclasts and thesubsequent deposition of the bisphosphonate in any newly formed bonenearby. In other embodiments, the bisphosphonate can be mixed with aprocessed allograft bone particle of the composite cement so that itprimarily resides at discrete locations in the graft and, in particular,on the surface of the graft particles. In this case, as opposed toincluding the bisphosphonate in the cement itself, the bisphosphonatewould be rapidly released and delivered to the local region as new boneforms in response to the bone grafting procedure. Any new bone, thusformed, would be infused with the bisphosphonate drug and would be morereadily preserved relative to normal bone. In yet other embodiments, thebisphosphonate can be mixed with both the cement powder and the boneparticles to obtain both of the beneficial effects of the approachesoutline above.

Further provided, in some embodiments of the presently-disclosed subjectmatter, are methods for treating a bone defect. In some embodiments, amethod for treating a bone defect is provided that comprises the stepsof: providing a bone graft composition of the presently-disclosedsubject matter; and administering an effective amount of the bone graftcomposition to a bone defect site in a subject.

As used herein, the terms “treatment” or “treating” relate to anytreatment of a bone defect, including, but not limited to, prophylactictreatment and therapeutic treatment. As such, the terms “treatment” or“treating” include, but are not limited to: preventing a bone defect orthe development of a bone defect; inhibiting the progression of a bonedefect; arresting or preventing the development of a bone defect;reducing the severity of a bone defect; ameliorating or relievingsymptoms associated with a bone defect; and causing a regression of thebone defect or one or more of the symptoms associated with the bonedefect.

The term “bone defect” is used herein to refer to any imperfection ordiscontinuity in the structure of a bone. For example, in someembodiments, the bone defect site is a bone void, or, in other words, anempty space that is typically occupied by bone. As another example, insome embodiments, the bone defect is a bone fracture or a break in thecontinuity of a bone. As yet another example, in some embodiments, thebone defect site is a site of an intended bone fusion, such as siteswhere portions of bone are rubbing against one another.

For administration of a bone graft composition disclosed herein, thebone graft compositions are typically administered in an amountsufficient to fill the site of the bone defect, i.e., an “effectiveamount.” Of course, the optimum amount of a bone graft composition usedto fill a bone defect will vary depending on the size and/or shape ofthe particular bone defect being filled. However, determination andadjustment of the amount of a bone graft composition to be used in aparticular application, as well as when and how to make suchadjustments, can be ascertained using only routine experimentation.

In some embodiments of the therapeutic methods described herein, thebone graft compositions can be used in association with various otherdevices commonly used to treat a bone defect. It has been observed thatthe placement of various devices into a bone defect often fails due tothe device becoming dislodged or otherwise removed from the bone or bonedefect. For example, screws are often placed into a bone, either aloneor in association with various cements, as shown in FIGS. 45A and 45B,respectively. However, the placement of a screw into a bone,particularly when the placement of screw into a bone is augmented withcement, frequently results in a failure of the cement composition,either at the interface of the bone and the cement, as shown in FIG.45C, or at the interface of the screw and the cement, as shown in FIG.45D. By using the bone graft compositions of the presently-disclosedsubject matter though, it has further been observed that the processedbone particles bridge across and strengthen both the screw-cement andcement-bone interfaces FIG. 45E. As such, in some embodiments of thepresently-disclosed methods for treating a bone defect, an effectiveamount of a bone graft composition can be administered to a site of abone defect in a subject prior to placing a device, such as a screw, inthe bone defect site.

In yet further embodiments of the presently-disclosed subject matter,kits are provided. In some embodiments, a kit is provided that includesa biologically-resorbable cement powder and a plurality of processedbone particles, where each of the processed bone particles has a shapeconfigured to interconnect with adjacent bone particles. In someembodiments of the kits, the biologically-resorbable cement powder andthe processed bone particles are packed in separate vessels or arepackaged together in a single vessel.

In some embodiments, the bone particles included in the kit arelyophilized or are otherwise dehydrated. In this regard, in someembodiments, the kit further includes an aqueous vehicle for adding tothe cement powder, the bone particles, or both the cement powder andbone particles. In some embodiments, the aqueous vehicle can be meteredand packaged in a separate vessel such that the vessel includes aprecise amount of aqueous vehicle for preparing a bone graft compositionhaving a desired consistency. In other embodiments, the kit furthercomprises instructions for mixing the cement powder and the boneparticles, and then combining that mixture with a prescribed amount ofan aqueous vehicle such that a bone graft composition having a desiredconsistency is produced and can then be administered to a subject.

As used herein, the term “subject” includes both human and animalsubjects. Thus, veterinary therapeutic uses are provided in accordancewith the presently disclosed subject matter. As such, thepresently-disclosed subject matter provides for the treatment of mammalssuch as humans, as well as those mammals of importance due to beingendangered, such as Siberian tigers; of economic importance, such asanimals raised on farms for consumption by humans; and/or animals ofsocial importance to humans, such as animals kept as pets or in zoos.Examples of such animals include but are not limited to: carnivores suchas cats and dogs; swine, including pigs, hogs, and wild boars; ruminantsand/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats,bison, and camels; and horses. Also provided is the treatment of birds,including the treatment of those kinds of birds that are endangeredand/or kept in zoos, as well as fowl, and more particularly domesticatedfowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guineafowl, and the like, as they are also of economic importance to humans.Thus, also provided is the treatment of livestock, including, but notlimited to, domesticated swine, ruminants, ungulates, horses (includingrace horses), poultry, and the like.

The presently-disclosed subject matter is further illustrated by thefollowing specific but non-limiting examples. Some of the followingexamples may include compilations of data that are representative ofdata gathered at various times during the course of development andexperimentation related to the present invention. Additionally, some ofthe following examples are prophetic, notwithstanding the numericalvalues, results and/or data referred to and contained in the examples.

EXAMPLES Example 1 Fabrication of Planar Cortical Bone Particles

To fabricate planar cortical bone particles through the refinement of anintact bone into a larger number of separate cortical bone particles, anintact bone was first obtained, and all of the soft tissue was removedfrom the diaphysis of the bone, with the larger pieces of soft tissuebeing removed using a blade, and the periosteum and connective tissuesbeing removed using a stainless steel wire wheel or brush. Next, thedistal and proximal ends of the bone were removed with a saw, leavingonly the diaphyseal region of the bone. All of the bone marrow and softtissue inside the bone's shaft was then physically removed, and the bonewas rinsed out. A reamer was used to remove any remaining cancellousbone material from the inside of the diaphyseal bone until only corticalbone remained. The resulting shaft of cortical bone was then thoroughlycleaned with detergent and deionized (DI) water, and the shaft ofcortical bone was subsequently soaked in 100 percent ethanol overnight.

After the overnight ethanol bath, each shaft of bone was then dried andcut longitudinally to create 3 to 4 shanks of cortical bone that wereapproximately 1 inch wide. Those fabricated pieces of cortical bone werecut with the longitudinal axis of the bone material running in the samedirection as the finished parts largest dimension. A surface grinder wasthen used to create thinned pieces of cortical bone that were 0.5 mmthick and approximately 1 to 2 inches square. That creation of thinnedpieces of cortical bone was done by machining small portions of eachbone shank one at a time due to the shanks' curved and organic shape.Once a 0.5 mm-thick piece of cortical bone stock was created, the bone'slongitudinal direction was then labeled, and the 0.5 mm-thick pieces ofbone stock were soaked in DI water under a vacuum for an hour. Thisallowed the material to soften or become slightly pliable and alsofilled all the open voids with water to minimize the amount ofsplintering in the subsequent stamping/punching process and to reducewaste. The 0.5 mm-thick bone stock was then removed from the DI waterand its surface blotted dry. The bone was then inserted in a custompunch with care taken to insure that the longitudinal direction of thebone was in the correct orientation in the punch. The punch was thenused to create a matrix of shaped cortical bone particles with a 0.5 mmthickness. A microscope with a scale was then used to measure theindividual bone pieces and ensure that their size and shape were inaccordance with the appropriate specifications. Typically, the corticalbone particles fabricated were 2.5 mm long and 1.5 mm wide by 0.5 mmthick.

Example 2 Fabrication of Complex Cortical Bone Particles

To fabricate more complex, three-dimensional cortical bone particles byrefining an intact bone into a large number of cortical bone particles,an intact bone was first obtained, and all of the soft tissue wasremoved from the diaphysis of the bone, with the larger pieces of softtissue being removed using a blade, and the periosteum and connectivetissues being removed using a stainless steel wire wheel or brush. Next,the distal and proximal ends of the bone were removed with a saw leavingonly the diaphyseal region of the bone. All the bone marrow and softtissue inside the bone's shaft was then physically removed, and the bonewas rinsed out. A reamer was used to remove any remaining cancellousbone material from the inside of the diaphyseal bone until only corticalbone remained. The resulting shaft of cortical bone was then thoroughlycleaned with detergent and dionized (DI) water, and the shaft ofcortical bone was subsequently soaked in 100 percent ethanol overnight.

Subsequent to the overnight ethanol bath, each shaft of bone was thendried and cut longitudinally to produce “matchstick” size shanks ofbone, with each shank being 4 to 5 mm across and approximately 75 mm inlength. Each of these “matchsticks” of cortical bone was then fixed in athimble-sized pot using an epoxy or polyester resin with care taken tobe sure that the “matchstick” pieces of bone potted concentric with thecenter of the pot. Once the bone material was fixed in a pot, a turningoperation was then used to create the “dumbbell” like shapes (see, e.g.,FIGS. 1A-1C). Briefly, each pot containing the bone material was mountedin a lathe, but, instead of a traditional cutter as is commonly used inlathe operations, a thin abrasive disk (approximately 0.5 mm thick) wasused to shape the bone particles as the abrasive disk preventedsplintering of the bone material. During the lathing procedure, theabrasive disk was used to cut the bone material perpendicular to therotation of the bone material in the lathe. Once the dumbbell-shapedbone particles were formed, a microscope was then used in conjunctionwith a scale to ensure the bone particles were of the appropriate sizeand dimensions. For most applications, dumbbell-shaped bone particleswere fabricated that were about 2.5 mm long, 1.5 mm diameter at the endsand 0.5 mm diameter in the center portion.

Example 3 Partial Demineralization of Cortical Bone

To partially demineralize the fabricated cortical bone particles byremoving the calcium from the surface of the particles, 9.0 to 10.0 g ofclean, defatted, cortical bone particles were first placed in a 1000 mlglass beaker with a magnetic stir bar. Approximately 600 ml of 0.05N HClwas then added to the container to ensure a sufficient volume of acidwas present to prevent neutralization during the demineralizationprocess. The acid solution containing the cortical bone particles wasthen stirred for 90 min using a magnetic stir bar to facilitate propermixing. After 90 min, the bone particles were then allowed to settle outin the solution, and the acid was poured off by using a thin mesh,stainless steel wire strainer. The bone pieces were then immediatelywashed with DI water until the pH was neutralized. Subsequently, thebone pieces were dried for several hours in a dehydrator/oven at atemperature of approximately 100° F. (38° C.). Further measurements ofthe decalcified layer, using a light microscope and a scanning electronmicroscope (SEM) to examine the surface morphology of the acid-soakedrods, revealed that the procedure resulted in approximately 10%decalcification of each piece of bone.

Example 4 Analysis of Bending Strength of Calcium Phosphate(Tetracalcium Phosphate/Monocalcium Phosphate/Calcium Carbonate)-basedBone Graft Composition

To assess the bending strength of a calcium phosphate cement comprisedof a mixture of tetracalcium phosphate, monocalcium phosphate, andcalcium carbonate powder and augmented with specially-shaped corticalbone particles, mechanical testing experiments using such a calciumphosphate cement with various amounts of partially-demineralized, shapedcortical bone particles were undertaken to determine the mechanicalbehavior effects of adding various amounts of the specially-processedcortical bone particles to the exemplar calcium-based cement. Briefly,in these experiments, a calcium phosphate cement consisting oftetracalcium phosphate (TTCP) powder, monocalcium phosphate (MCP)powder, and calcium carbonate powder was used and was prepared by mixingthe powder mixture with water in a 2.6:1 powder to water ratio by weightto form a paste, which was then capable of setting within fifteenminutes into a solid mass.

Similar to the methodology described herein above, cortical boneparticles were then created through a machining process that yieldedspecially-shaped particles approximately 2.5 mm in length, 1.5 mm inwidth at each end, and 0.5 mm thickness on average with a 0.5 mm widecentral portion (see, e.g., FIG. 1A). These bone particles were obtainedfrom the diaphyseal regions of porcine femora and tibae and werepartially demineralized using the methodology described above. After theparticles were formed and dehydrated, they were then weighed and addedto the premixed cement powder in volume ratios of 10.0% and 20.0%. Thedry components of cement powders and dry bone particles were thenthoroughly mixed.

For purposes of comparison, calcium phosphate mixtures were also madeusing non-specially shaped cortical bone particles of similar size tothose described above. The non-specially shaped particles were producedby grinding 0.5 mm thick scrap bone that was produced during thefabrication of the shaped bone particles. The scrap bone was ground in arotary mill to produce a variety of particle sizes that were sortedusing two sieves over multiple passes to produce particles between 1.75mm and 0.5 mm particle size dimensions. These particles were alsopartially demineralized as described above and dehydrated before beingadded to the calcium phosphate powder, and were thoroughly mixed toproduce approximate volume ratios of 10% and 40%.

Subsequent to the formation of the powders and the bone particles, testsamples were then created by combining the dry bone and cement materialswith an appropriate amount of distilled water (2.6:1 ratio of calciumphosphate powder to water by weight), mixing with a thin metal spatulauntil a consistent wet paste was formed. Next, the materials were spreadinto cylindrical Teflon® (Du Pont de Nemours and Company Corporation,Wilmington, Del.) molds that were designed to create test samples 20 mmin length with a diameter of 8 mm.

The bending test was then performed using a three-point bending fixturewith an 11 mm support span and a central radiused “blade” that applied aload to the top of the horizontally oriented specimen at a rate of 0.1in/min (2.54 mm/min), as depicted in FIG. 3. The maximum force achievedprior to the first failure of the sample resulting in a 40% reduction inapplied load or a significant change in specimen stiffness was recorded.The failure stress was calculated as the flexural strength based on theapplied bending load and the sample geometry using the formula σ=Mc/I,where I is the area moment of inertia of the circular cross-section ofthe sample.

TABLE 1 Bending Strength (MPa) of Calcium Phosphate (TetracalciumPhosphate/Monocalcium Phosphate/Calcium Carbonate)-Based Bone GraftCompositions CaP 100% Tr 10% Tr 20% Alt 10% Alt 40% Mean 4.4 8.1 6.4 7.93.5 Std. Dev. 1.3 1.9 1.1 2.2 0.9 P <0.005 <0.01 <0.005 NS

As shown in FIG. 4 and Table 1, upon analysis of the results from theseexperiments, it was observed that adding specially-shaped and partiallydemineralized bone particles (Tr) to a calcium phosphate cementsignificantly improve the mechanical properties of the cement in thedemanding loading mode of bending. In other words, the foregoing resultsdemonstrated that an osteoconductive, remodelable, resorbable, andosteoinductive material can be added to a calcium phosphate cement andcan be used to increase the bending strength of the calcium phosphatecement. In contrast, when the bone particles added at 40% werenon-specially shaped (Alt), the bending strength decreasedsignificantly, as also shown in FIG. 4, thus further indicating that themechanical behavior of bone graft compositions can be improved by theinclusion of the specially-shaped bone particles. Additionally, it wasfound that a higher percentage of non-specially shaped bone particleswas required to be added to the cement to achieve a similar level ofremodeling potential through interconnectedness as compared to thespecially-shaped particles that were included at a lower percentage.

Example 5 Analysis of Bending Strength of Calcium Phosphate(Alpha-Tricalcium Phosphate/Hydroxyapatite)-based Bone Graft Composition

To further assess the bending strength of calcium phosphate-basedcements augmented with specially-shaped cortical bone particles,additional mechanical testing experiments using an alternative calciumphosphate cement with various amounts of partially-demineralized,specially-shaped cortical bone particles were undertaken. Briefly, inthis further experiment, a commercial-grade calcium phosphate cement,including an alpha-tricalcium phosphate powder that, upon mixing with asetting solution, formed precipitated hydroxyapatite, was used. Whenthis powder mixture was mixed with water in a 2.6:1 powder-to-waterratio by weight, a paste formed that was then capable of setting withinfifteen minutes into a solid mass.

Similar to the methodology described above, cortical bone particles werethen again created through a machining process that yieldedspecially-shaped particles approximately 2.5 mm in length, 1.5 mm inwidth at each end, and 0.5 mm in thickness, on average, with a 0.5 mmwide central region (see, e.g., FIG. 1A). These bone particles were onceagain obtained from the diaphyseal regions of porcine femora and tibaeand were partially demineralized subsequent to weighing and adding thebone particles to the premixed cement powder in a volume ratio of 10.0%.The dry components of cement powders and dry bone particles were thenthoroughly mixed.

Again, for purposes of comparison, calcium phosphate mixtures were alsomade using non-specially shaped cortical bone particles of similar sizeto those described above. The non-specially shaped particles were againproduced using scrap bone and had particle size dimensions of between1.75 mm and 0.5 mm. These particles were also partially demineralized asdescribed above and dehydrated before being added to the calciumphosphate powder and thoroughly mixed to produce an approximate volumeratio 40%, as it was found in the experiments described above that ahigher percentage of non-specially shaped bone particles was required tobe added to the cement to achieve a similar level of remodelingpotential through interconnectedness as compared to the specially-shapedbone particles included at a lower percentage.

Subsequent to the formation of the powders and the bone particles, testsamples were once again created by combining the dry bone and cementmaterials with an appropriate amount of distilled water (2.6:1 ratio ofcalcium phosphate powder to water by weight), and mixing with a thinmetal spatula until a consistent wet paste was formed. The materialswere then again spread into cylindrical Teflon® (Du Pont De Nemours AndCompany Corporation, Wilmington, Del.) molds to yield test samples 20 mmin length with a diameter of 8 mm.

The bending test was again performed with these additional calciumphosphate-based samples using the three-point bending fixture describedabove (see FIG. 3). The maximum force achieved prior to the firstfailure of the sample resulting in a 40% reduction in applied load or asignificant change in specimen stiffness was once more recorded, and thefailure stress for these samples was subsequently calculated as theflexural strength based on the applied bending load and the samplegeometry using the formula σ=Mc/I, where I is the area moment of inertiaof the circular cross-section of the sample.

TABLE 2 Bending Strength (MPa) of Calcium Phosphate (Alpha-TricalciumPhosphate/Precipitated Hydroxyapatite)-Based Bone Graft CompositionsCaP* 100% Tr 10% Alt 40% Mean 3.8 4.2 2.1 Std. Dev. 1.4 1.2 0.9 P NS<0.005

As shown in FIG. 5 and Table 2, upon analysis of the results from theseexperiments, it was once more observed that adding specially-shaped andpartially demineralized bone particles (Tr) to an alternative calciumphosphate cement, which was comprised of alpha-tricalciumphosphate/precipitated hydroxyapatite, significantly improved themechanical properties of the cement in the demanding loading mode ofbending. Similar to the experiment described above in Example 4, whenthe bone particles added at 40% were non-specially shaped (Alt), thebending strength decreased significantly, as shown in FIG. 5, thusfurther indicating that the bending strength of the calcium phosphatecould be increased by utilizing specially-shaped bone particles.

Example 6 Analysis of Bending Strength of Calcium Sulfate-based BoneGraft Composition

To assess the bending strength of a non-calcium phosphate-based cementin conjunction with the presently-described, specially-shaped corticalbone particles, mechanical testing experiments using a calcium sulfate(CaS) cement with various amounts of partially-demineralized,specially-shaped cortical bone particles were also undertaken. Briefly,in this further experiment, a commercial grade CaS cement was used andwas mixed with water in a 2:1 powder to water ratio by weight, such thata paste was formed that was capable of setting within twenty minutesinto a solid mass.

Similar to the methodology described above, cortical bone particles werethen again created through a machining process that yieldedspecially-shaped particles approximately 2.5 mm in length, 1.5 mm inwidth at each end, and 0.5 mm thickness on average with a 0.5 mm widecentral region (see, e.g., FIG. 1A). These bone particles were alsoobtained from the diaphyseal regions of porcine femora and tibae andwere partially demineralized subsequent to weighing and adding the boneparticles to the premixed cement powder in a volume ratios of 10.0% and20.0%. The dry components of cement powders and dry bone particles werethen thoroughly mixed.

Again, for purposes of comparison, CaS mixtures were also made usingnon-specially shaped cortical bone particles of similar size to thosedescribed above. The non-specially shaped particles were again producedusing scrap bone and had particle size dimensions of between 1.75 mm and0.5 mm. These particles were also partially demineralized as describedabove and dehydrated before being added to the calcium phosphate powder,and were thoroughly mixed to produce approximate volume ratios of 10%and 40%, as it was found in the experiments described above that ahigher percentage of non-specially shaped bone particles was required tobe added to the cement to achieve a similar level of remodelingpotential through interconnectedness as compared to the specially-shapedparticles that were included at a lower percentage.

Subsequent to the formation of the powders and the bone particles, testsamples were once again created by combining the dry bone and cementmaterials with an appropriate amount of distilled water (2:1 ratio ofCaS powder to water by weight), and mixing with a thin metal spatulauntil a consistent wet paste was formed. The materials were then againspread into cylindrical Teflon® (Du Pont de Nemours and CompanyCorporation, Wilmington, Del.) molds to create test samples 20 mm inlength with a diameter of 8 mm.

The bending test was then again performed using the three-point bendingfixture described above (see FIG. 3). The maximum force achieved priorto the first failure of the sample resulting in a 40% reduction inapplied load or a significant change in specimen stiffness was once morerecorded, and the failure stress for these samples was subsequentlycalculated as the flexural strength based on the applied bending loadand the sample geometry using the formula σ=Mc/I, where I is the areamoment of inertia of the circular cross-section of the sample.

TABLE 3 Bending Strength (MPa) of Calcium Sulfate Based CompositeCements CaS 100% Tr 10% Tr 20% Alt 10% Alt 40% Mean 3.7 5.0 5.0 3.0 1.9Std. Dev. 0.4 0.9 0.9 0.5 0.1 P <0.005 <0.005 <0.05 <0.0005

As shown in FIG. 6 and Table 3, upon analysis of the results from theseexperiments, it was observed that the addition of specially-shaped andpartially demineralized bone particles (Tr) to a different calcium-basedcement, namely a CaS cement, also significantly improved the mechanicalproperties of the cement in the demanding loading mode of bending,demonstrating that an osteoconductive, remodelable, resorbable, andosteoinductive material can be added to a CaS cement and used toincrease the bending strength of the CaS cement by utilizing boneparticles having specialized shapes. Similar to the experiment describedabove in Examples 4 and 5, when the bone particles added at 10% and 40%were non-specially shaped (Alt), the bending strength decreasedsignificantly, as shown in FIG. 6.

Example 7 Analysis of Bending Strength of Calcium Sulfate-based BoneGraft Composition Including Elongated Cortical Bone Particles

To assess the bending strength of a calcium-based cement augmented withspecially-shaped cortical bone particles having a different shape thanthose described above, mechanical testing experiments were undertakenusing calcium sulfate (CaS) cement with a ten percent (10%) amount ofpartially-demineralized cortical bone particles that were shaped in anelongated fashion, which was designed to mimic a chain of independent,discrete supporting structures. In these experiments, commerciallyavailable CaS powders were mixed with water in a 2:1 powder to waterratio by weight, such that a paste was formed that was capable ofsetting within twenty minutes into a solid mass. Unlike the methodologydescribed above, the cortical bone particles in these experiments werecreated through the machining process to yield specially-shapedparticles that were approximately 10 or 14.5 mm in length, 1.5 mm inwidth at each end, and 0.5 mm thickness on average with a 0.5 mm widecenter portion (FIG. 1G). These bone particles were also obtained fromthe diaphyseal regions of porcine femora and tibae and were partiallydemineralized using the methodology described above such that a flexibleelongated structure was created. After the particles were formed anddehydrated, the particles were then weighed and added to the premixedcement powder in a 10% volume ratio. The dry components of cementpowders and dry bone particles were then thoroughly mixed. Forcomparison purposes, a 100% CaS cement control was also created usingthe same described powder and water in a 2:1 ratio by weight.

Subsequent to the formation of the powders and the bone particles, testsamples were then created by combining the dry materials with theappropriate amount of distilled water (2:1 ratio of CaS powder to waterby weight), and mixing with a spatula until a consistent wet paste wasformed. Next, the materials were spread into cylindrical Teflon® (DuPont de Nemours and Company Corporation, Wilmington, Del.) moldsdesigned to create test samples 20 mm in length with a diameter of 8 mm.

Similar to the experiments described above, the bending test was thenperformed using a three-point bending fixture with an 11 mm support spanand a central radiused “blade” that applied a load to the top of thehorizontally oriented specimen at a rate of 0.1 in/min (2.54 mm/min).The maximum force achieved prior to the first failure of the sampleresulting in a 40% reduction in applied load or a significant change inspecimen stiffness was again recorded for these samples, and the failurestress was calculated as the flexural strength based on the appliedbending load and the sample geometry using the formula σ=Mc/I, where Iis the area moment of inertia of the circular cross-section of thesample.

The bending strength test results obtained from these experiments aredepicted in FIG. 7 and in Table 4 below, where Table 4 contains themaximum force data recorded for each test during the 3-point Bendingtest.

TABLE 4 Bending Strength (MPa) of Calcium Sulfate Based CompositeCements Including Elongated Cortical Bone Particles. Elong CaS 100% 10%Mean 3.7 6.0 Std. Dev. 0.4 1.9 P <0.005

Once more, the results indicated that adding specially-shaped andpartially demineralized bone particles (Elong) to a CaS cementsignificantly improved its mechanical properties in the demandingloading mode of bending, and further indicating that the mechanicalbehavior of the bone graft compositions can be improved via the presenceof the specially-shaped bone particles.

Example 8 Analysis of Shear Strength of Calcium Phosphate (TetracalciumPhosphate/Monocalcium Phosphate/Calcium Carbonate)-based Bone GraftComposition

To assess the shear strength of a calcium phosphate cement comprised ofa mixture of tetracalcium phosphate, monocalcium phosphate, and calciumcarbonate powder and augmented with specially-shaped cortical boneparticles, mechanical testing experiments using such a calcium phosphatecement with various amounts of partially-demineralized, shaped corticalbone particles were also undertaken. These experiments were undertaken,at least in part, because it was believed that the mechanical propertiesin the demanding loading mode of shear is also important for themechanical function of the materials when implanted in the body forcontrol of fracture fragments. Briefly, in these experiments, a calciumphosphate cement consisting of tetracalcium phosphate (TTCP) powder,monocalcium phosphate (MCP) powder, and calcium carbonate powder wasused, and was prepared by mixing the powder mixture with water in a2.6:1 powder-to-water ratio by weight to form a paste, which was thencapable of setting within fifteen minutes into a solid mass.

Similar to the methodology described above, cortical bone particles werethen created through a machining process that yielded specially-shapedparticles approximately 2.5 mm in length, 1.5 mm in width at each end,and 0.5 mm thickness on average with a 0.5 mm wide center portion (see,e.g., FIG. 1A). These bone particles were obtained from the diaphysealregions of porcine femora and tibae and were partially demineralizedusing the methodology described above. After the particles were formedand dehydrated, they were then weighed and added to the premixed cementpowder in volume ratios of 10.0% and 20.0%. The dry components of cementpowders and dry bone particles were then thoroughly mixed.

For purposes of comparison, calcium phosphate mixtures were also madeusing non-specially shaped cortical bone particles of similar size tothose described above. Again, the non-specially shaped particles wereproduced by grinding 0.5 mm thick scrap bone to produce particlesbetween 1.75 mm and 0.5 mm particle size dimensions. These particleswere also partially demineralized as described above and dehydratedbefore being added to the calcium phosphate powder and thoroughly mixedto produce approximate volume ratios of 10% and 40%%, as it was found inthe experiments described above that a higher percentage ofnon-specially shaped bone particles was required to be added to thecement to achieve a similar level of remodeling potential throughinterconnectedness as compared to the specially-shaped particles thatwere included at a lower percentage.

Subsequent to the formation of the powders and the bone particles, testsamples were then created by combining the dry bone and cement materialswith an appropriate amount of distilled water (2.6:1 ratio of calciumphosphate powder to water by weight), mixing with a thin metal spatula,until a consistent wet paste was formed. Next, the materials were spreadinto tapered cylindrical (conical) molds that had been machined into analuminum block. The molds were designed to create test samples 20 mm inlength with an average diameter of 8 mm spread over a 1-degree taperalong the length of the sample. Thus, the minimum diameter of the testsample was 7.83 mm, and the maximum diameter was 8.17 mm. The rationalefor the taper was for ease of removal of the samples from the one-piecemolds and for shear testing using a custom made shear test fixture thatshared the same taper as the molds, as depicted in FIG. 8.

TABLE 5 Shear Strength (MPa) of Calcium Phosphate Based CompositeCements CaP 100% Tr 10% Tr 20% Alt 10% Alt 40% Mean 4.3 3.4 2.3 3.0 1.6Std. Dev. 0.7 0.5 0.4 0.7 0.4 p <0.005 <0.0001 <0.001 <0.00001

As shown in FIG. 9 and Table 5, upon analysis of the results from theseexperiments, it was observed that adding specially-shaped and partiallydemineralized bone particles (Tr) to a calcium phosphate cement at a 10%volume fraction improved the mechanical properties of the composition inthe demanding loading mode of shear, while the addition of 20%specially-shaped bone particles and both 10% and 40% non-speciallyshaped bone particles (Alt) significantly weakened the calcium phosphatein shear. As such, these results thus indicated that an osteoconductive,remodelable, resorbable, and osteoinductive material can be added to acalcium phosphate cement and improve its mechanical shearing properties.

Example 9 Analysis of Bending Strength of Calcium Phosphate(Alpha-Tricalcium Phosphate/Hydroxyapatite)-based Bone Graft Composition

To further assess the shear strength of calcium phosphate-based cementsaugmented with specially-shaped cortical bone particles, additionalmechanical testing experiments using an alternative calcium phosphatecement with various amounts of partially-demineralized, shaped corticalbone particles were undertaken. Briefly, in these further experiments, acalcium phosphate cement comprising a commercial grade calcium phosphatecement, including alpha-tricalcium phosphate powder that, upon mixingwith a setting solution forms precipitated hydroxyapatite, was used.When this powder mixture was mixed with water in a 2.6:1 powder-to-waterratio by weight, a paste formed that was capable of setting withinfifteen minutes into a solid mass.

Similar to the methodology described above, cortical bone particles werethen again created through a machining process that yielded speciallyshaped particles approximately 2.5 mm in length, 1.5 mm in width at eachend, and 0.5 mm thickness on average with a 0.5 mm wide center portion(see, e.g., FIG. 1A). These bone particles were once again obtained fromthe diaphyseal regions of porcine femora and tibae and were partiallydemineralized subsequent to weighing and adding the bone particles tothe premixed cement powder in volume ratios of 10.0% and 20%. The drycomponents of cement powders and dry bone particles were then thoroughlymixed.

Again, for purposes of comparison, calcium phosphate mixtures were alsomade using non-specially shaped cortical bone particles of similar sizeto those described above. The non-specially shaped particles were againproduced using scrap bone and had particle size dimensions of between1.75 mm and 0.5 mm. These particles were also partially demineralized asdescribed above and dehydrated before being added to the calciumphosphate powder, and were thoroughly mixed to produce an approximatevolume ratio 40%%, as it was found in the experiments described abovethat a higher percentage of non-specially shaped bone particles wasrequired to be added to the cement to achieve a similar level ofremodeling potential through interconnectedness as compared to thespecially-shaped particles that were included at a lower percentage.

Subsequent to the formation of the powders and the bone particles, testsamples were once again created by combining the dry bone and cementmaterials with an appropriate amount of distilled water (2.6:1 ratio ofcalcium phosphate powder to water by weight), mixing with a thin metalspatula, until a consistent wet paste was formed. Next, the materialswere once again spread into tapered cylindrical (conical) molds that hadbeen machined into an aluminum block. The molds were designed to createtest samples 20 mm in length with an average diameter of 8 mm spreadover a 1-degree taper along the length of the sample. Thus, the minimumdiameter of the test sample was 7.83 mm, and the maximum diameter was8.17 mm. The rationale for the taper was for ease of removal of thesamples from the one-piece molds and for shear testing using a custommade shear test fixture, depicted in FIG. 8, that shared the same taperas the molds.

TABLE 6 Shear Strength (MPa) of Calcium Phosphate Based CompositeCements CaP 100% Tr 10% Tr 20% Alt 40% Mean 2.8 3.2 1.5 1.2 Std. Dev.0.4 0.7 0.3 0.4 p NS <0.00001 <0.00001

As shown in FIG. 10 and Table 6, upon analysis of the results from theseexperiments, it was observed that adding specially-shaped and partiallydemineralized bone particles (Tr) to a further calcium phosphate cementat a 10% volume fraction improved the mechanical properties of thecomposition in the demanding loading mode of shear, while the additionof 20% specially-shaped bone particles and 40% non-specially shaped boneparticles (Alt) significantly weakened the calcium phosphate in shear.As such, these results thus also indicated that an osteoconductive,remodelable, resorbable, and osteoinductive material can be added to acalcium phosphate cement and improve its mechanical shearing properties.

Example 10 Analysis of Shear Strength of a Calcium Sulfate-based BoneGraft Composition

To assess the shear strength of a non-calcium phosphate-based cement inconjunction with the presently-described, specially-shaped cortical boneparticles, mechanical testing experiments using a calcium sulfate (CaS)cement with various amounts of partially-demineralized, shaped corticalbone particles were also undertaken to assess the mechanical behavioreffects of adding various percentages of the specially processedcortical bone particles to that calcium-based cement. Briefly, in thisfurther experiment, a commercial grade CaS cement was used and was mixedwith water in a 2:1 powder-to-water ratio by weight, such that a pastewas formed that was capable of setting within twenty minutes into asolid mass.

Similar to the methodology described above, cortical bone particles werethen again created through a machining process that yieldedspecially-shaped particles approximately 2.5 mm in length, 1.5 mm inwidth at each end, and 0.5 mm thickness on average with a 0.5 mm widecenter portion (see, e.g., FIG. 1A). These bone particles were alsoobtained from the diaphyseal regions of porcine femora and tibae andwere partially demineralized subsequent to weighing and adding the boneparticles to the premixed cement powder in a volume ratios of 10.0% and20.0%. The dry components of cement powders and dry bone particles werethen thoroughly mixed.

Subsequent to the formation of the powders and the bone particles, testsamples were once again created by combining the dry bone and cementmaterials with an appropriate amount of distilled water (2:1 ratio ofCaS powder-to-water by weight), and mixing with a thin metal spatulauntil a consistent wet paste was formed. Next, the materials were onceagain spread into tapered cylindrical (conical) molds that had beenmachined into an aluminum block. The molds were designed to create testsamples 20 mm in length with an average diameter of 8 mm spread over a1-degree taper along the length of the sample. Thus, the minimumdiameter of the test sample was 7.83 mm, and the maximum diameter was8.17 mm. The rationale for the taper was for ease of removal of thesamples from the one-piece molds and for shear testing using a custommade shear test fixture, depicted in FIG. 8, that shared the same taperas the molds.

TABLE 7 Shear Strength (MPa) of Calcium Phosphate Based CompositeCements CaP 100% Tr 10% Tr 20% Mean 0.8 1.0 0.9 Std. Dev. 0.4 0.5 0.4

As shown in FIG. 11 and Table 7, upon analysis of the results from theseexperiments, it was observed that the addition of specially-shaped andpartially demineralized bone particles (Tr) to a CaS cement did notsignificantly compromise its mechanical properties in the demandingloading mode of shear. In other words, the foregoing results furtherdemonstrate that an osteoconductive, remodelable, resorbable, andosteoinductive material can be added to a CaS cement without negativelyaffecting its mechanical properties, provided the particles possess theshapes tested here.

Example 11 Diametral Tensile Strength of Calcium Sulfate-based BoneGraft Composition

To assess the diametral tensile strength of a calcium-sulfate cementincorporating the specially-shaped bone particles of thepresently-disclosed subject matter, mechanical testing experiments usinga calcium sulfate (CaS) cement that incorporated various amounts ofpartially demineralized shaped cortical bone particles were undertaken.In these experiments, and similar to the experiments described above, acommercial grade CaS cement was used that, when mixed with water in a2:1 powder-to-water ratio by weight, was capable of forming a paste thatset within twenty minutes into a solid mass. Also, in these experimentsand again similar to the methodology described above, cortical boneparticles were created through a machining process that yieldedspecially-shaped particles approximately 2.5 mm in length, 1.5 mm inwidth at each end, and 0.5 mm thickness on average with a 0.5 mm widecenter portion (FIG. 1A). These bone particles were also obtained fromthe diaphyseal regions of porcine femora and tibae and were alsopartially demineralized using the methodology described above. After theparticles were formed and dehydrated, they were then weighed and addedto the premixed cement powder in volume ratios of 10.0% and 20.0%. Thedry components of cement powders and dry bone particles were thoroughlymixed.

For comparison, alternative mixtures were made using non-speciallyshaped cortical bone particles of similar size to those described above.The non-specially shaped particles were again produced by grinding 0.5mm thick scrap to produce particles between 1.75 mm and 0.5 mm particlesize dimensions, and were also partially demineralized as describedabove and dehydrated before being added to the CaS powder and thoroughlymixed to produce approximate volume ratios of 10% and 40%, as it wasfound in the experiments described above that a higher percentage ofnon-specially shaped bone particles was required to be added to thecement to achieve a similar level of remodeling potential throughinterconnectedness as compared to the specially-shaped particles thatwere included at a lower percentage.

The test samples were then created by combining the dry materials withan appropriate amount of distilled water (2:1 ratio of CaSpowder-to-water by weight), and mixing with a thin metal spatula until aconsistent wet paste was formed. Next, the materials were spread intotapered cylindrical (i.e., conical) molds that had been machined into analuminum block. The molds were designed to create test samples 20 mm inlength with a diameter of 8 mm.

The diametral tension test was performed using two polished parallelplatens (see FIG. 12). The cylindrical specimen was placed horizontallysuch that a compressive load could be applied in a directionperpendicular to the longitudinal axis at a rate of 0.1 in/min (2.54mm/min). The maximum force achieved prior to the first failure of thesample resulting in a 40% reduction in applied load or a significantchange in specimen stiffness was then recorded, and the failure stresswas calculated based on the applied load and the sample geometry usingthe formula σ=2P/(π*D*L), where P is the load, D is the diameter, and Lis the length of the specimen.

TABLE 8 Diametral Tensile Strength (MPa) of Calcium Sulfate BasedComposite Cements. CaS 100% Tr 10% Tr 20% Alt 10% Alt 40% Mean 1.1 1.51.4 0.9 0.7 Std. Dev. 0.2 0.4 0.4 0.2 0.2

Upon analysis of the results from the diametral tensile strengthexperiments, and as shown in FIG. 13 and Table 8, it was observed thatadding specially-shaped and partially demineralized bone particles (Tr)to a CaS cement can significantly increase its mechanical strength inthe demanding loading mode of diametral tension, while the addition of10% and 40% non-specially shaped bone particles (Alt) significantlyweakened the diametral tensile strength of the calcium phosphate. Assuch, these results also provide further support for the finding that anosteoconductive, remodelable, resorbable, and osteoinductive materialcan be added to a Ca-based cement and can be used improve its mechanicalproperties, provided the particles possess the unique shapes testedhere.

Example 12 Bending Toughness (Energy-to-Failure) of CalciumPhosphate-Based Bone Graft Composition when Loaded Dynamically

To assess the bending toughness of a calcium phosphate-based cementincorporating the specially-shaped bone particles of thepresently-disclosed subject matter when such a composition is loadeddynamically, mechanical testing experiments using calcium phosphatecement with various amounts of partially-demineralized, specially-shapedcortical bone particles were undertaken. Briefly, a calcium phosphatecement consisting of tetracalcium phosphate (TTCP) powder, monocalciumphosphate (MCP) powder, calcium carbonate powder was used, and was mixedwith water in a 2.6:1 powder-to-water ratio by weight, such that a pastewas formed that set within fifteen minutes into a solid mass.

Similar to the methodology described above, cortical bone particles fromthe diaphyseal regions of porcine femora and tibae were again createdthrough a machining process that yielded specially-shaped particlesapproximately 2.5 mm in length, 1.5 mm in width at each end, and 0.5 mmthickness on average with a 0.5 mm wide central region (FIG. 1A). Thesebone particles were then partially demineralized using the methodologydescribed above and, after the particles were formed and dehydrated,were then weighed and added to the premixed cement powder in volumeratios of approximately 10.0% and 20.0%. The dry components of cementpowders and dry bone particles were then thoroughly mixed.

Test samples were then created by combining the dry materials with anappropriate amount of distilled water (2.6:1 ratio of calcium phosphatepowder to water by weight), mixing with a thin metal spatula, until aconsistent wet paste was formed. Next, the materials were spread intocylindrical Teflon® (Du Pont De Nemours And Company Corporation,Wilmington, Del.) molds. The molds are designed to create test samples20 mm in length with a diameter of 8 mm.

Similar to experiments described above, the dynamic bending test wasperformed using a three-point bending fixture with an 11 mm support spanand a central radiused “blade” that applies a load to the top of thehorizontally oriented specimen at a rate of 10 mm/s. The toughness ofthe samples was calculated as the energy-to-failure by measuring theunder the load-displacement curve during the test to failure in bending.

TABLE 9 Bending Toughness (energy-to-failure: N-mm) of Calcium PhosphateBased Composite Cements. CaP 100% Tr 10% Mean 21.9 160.4 Std. Dev. 3.646.3 P <0.00001

Upon analysis of the results from the dynamic bending experiments, andas shown in Table 9, it was observed that the addition ofspecially-shaped and partially-demineralized bone particles to a calciumphosphate cement significantly improved its mechanical properties in thedemanding loading mode of bending, indicating that an osteoconductive,remodelable, resorbable, and osteoinductive material can effectively beadded to a calcium phosphate cement while significantly increasing itsbending toughness, provided the particles possess the unique shapestested here.

Example 13 Mechanical Testing of Calcium Phosphate Cement with VariousPercentage Inclusion of Partially Demineralized Elongated Cortical BoneParticles

Mechanical testing experiments using calcium phosphate cement withvarious amounts of partially demineralized elongated cortical boneparticles were undertaken to determine the mechanical behavior effectsof adding various percentages of specially processed cortical boneparticles to an exemplar calcium phosphate cement. Briefly, a commercialgrade calcium phosphate cement consisting of equimolar parts oftetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA)powders was used. When these powders were mixed with a solution ofsodium phosphate, a paste formed that set within twenty minutes into asolid hydroxyapatite ceramic mass.

Similar to the methodology described above, cortical bone particles werecreated through a milling process that yielded elongated particlesapproximately 5 mm in length and 0.5 mm thickness on average. These boneparticles were obtained from the mid-diaphyseal regions of porcinefemora and tibae and were also partially demineralized using themethodology described above. After the particles were formed and dried,the particles were then weighed and added to the premixed cement powderin the following weight ratios: 0.0%, 1.25%, 2.5%, 3.75%, and 5.0percent. The resulting cements were labeled X000, X125, X250, X375, andX500, respectively. Because the ceramic was denser than the partiallydemineralized bone particles, the volume percentages of bone in thecement were approximately 50% greater than the weight percentages. Thedry components of cement powders and dry bone particles were thoroughlymixed.

Test samples were then created by mixing the dry materials with anappropriate amount of a sodium phosphate (NaP) solution, using a thinmetal spatula, until a consistent wet paste was formed. Next, thematerials were spread into tapered cylindrical (conical) molds that hadbeen machined into an aluminum block. The molds were designed to createtest samples 20 mm in length with an average diameter of 8 mm spreadover a 1 degree taper along the length of the sample. Thus, the minimumdiameter of the test sample was 7.83 mm and the maximum diameter was8.17 mm. The rationale for the taper was for ease of removal of thesamples from the one-piece molds and for the shear testing.

Shear testing was done using a custom-made shear test fixture (see FIG.8). Three-point bending testing was done using the same sample type withthe maximum bending stress placed at the central point (8 mm diameter)of the sample. In both cases, however, the tests were performed byadvancing the fixture. Furthermore, and in addition to testing thestrength of each sample (bending and shear) that was measured as afunction of the sample geometry and the maximum load attained beforefailure, the work of failure (or toughness) of each sample wasdetermined by calculating the area under the load displacement curve foreach test.

The bending test results for these samples are depicted in FIG. 14. Thebending test results for a control calcium phosphate cement were similarto other published values for standard calcium phosphate cement (Xu etal., J Biomed Mater Res. 2001 Dec. 5; 57(3):457-66). The strength forthe 0% samples was 12.4±2.9 MPa (mean±SD). None of the samples withprocessed bone added were significantly different from the control(p>0.05). The strongest of the composites was the 3.75% bone materialwhich had a strength of 10.5±2.3 MPa. Table 10 below contains themaximum force data recorded for each test during the bending test, andit was the maximum force that was used to calculate the strength of eachtest sample

TABLE 10 3-Point Bending. XBC + XBC + XBC + XBC + XBC + Bone Source 0.0%1.25% 2.50% 3.75% 5.0% 45.16 279.24 192.85 156.56 152.29 218.43 40.64297.13 94.68 125.66 236.67 214.48 23 212.93 157.43 92.75 145.69 154.1551.09 203.39 172 220.79 163.45 173.65 48.35 215.5 166.62 243.51 219.3180.09 50.56 150.22 152.41 306.6 233.06 206.39 Bsource X000 X125 X250X375 X500 Mean 43.133 226.402 155.998 190.978 191.745 174.532 SD 10.59653.718 33.172 80.146 42.340 52.675

The bending toughness test results are depicted in FIG. 15 and in Table11 below. The bending toughness results showed greater toughness in thehigher percentage composites compared to the control cement.

TABLE 11 3-Point Bending Toughness. XBC + XBC + XBC + XBC + XBC + BoneSource 0.0% 1.25% 2.50% 3.75% 5.0% 15.897 16.893 12.058 10.532 36.56987.730 6.487 18.711 4.057 15.042 50.082 43.205 1.998 10.250 7.297 3.97130.344 21.818 4.271 11.095 10.562 133.208 19.176 28.934 4.651 12.7929.001 102.157 64.465 8.095 5.112 5.976 8.170 141.448 58.247 51.019Bsource X000 X125 X250 X375 X500 Mean 6.403 12.620 8.524 67.727 43.14740.134 SD 4.875 4.636 2.773 64.838 17.392 27.859

The shear strength test results are depicted in FIG. 16 and in Table 12below. Table 3 contains the maximum force data recorded for each testduring the shear test. It was that maximum force that was divided by thesample cross-sectional area and used to calculate the shear strength ofeach test sample. The shear strength of the control cement wassignificantly greater than the composited cements at all percentages,but each of these was reasonably strong in shear and none weresignificantly different from each other.

TABLE 12 Shear Strength. XBC + XBC + XBC + XBC + XBC + Bone Source 0.0%1.25% 2.50% 3.75% 5.0% 74.1 416.9 228.1 89.9 202.1 144.9 49.7 499.1177.7 210.7 284.2 132 44.7 473.2 236.5 131.1 165.2 220.5 32.1 435.3228.2 277 224.7 87.5 35.2 455.8 231.1 153 213.5 60.8 134.9 173.2 222.8179.5 182.3 179.4 329.8 Bsource X000 X125 X250 X375 X500 Mean 61.783408.917 220.733 173.533 212 137.516 SD 38.790 118.984 21.551 65.32841.344 58.502

The shear toughness test results are depicted in FIG. 17 and in Table 13below. The shear toughness showed no significant difference between anyof the sample groups.

TABLE 13 Shear Toughness. XBC + XBC + XBC + XBC + XBC + Bone Source 0.0%1.25% 2.50% 3.75% 5.0% 7.2 17.4 20.3 9 31.2 15.9 7.1 25 27.8 15.7 16.413.8 3.5 31 12.7 14 16.4 19.5 3.2 19.7 15.6 20.1 23 13.2 3.8 18.6 20.713.6 19 9.7 12.5 18.4 21.7 14 14.7 22.2 24.9 Bsource X000 X125 X250 X375X500 Mean 6.217 21.683 19.8 14.4 20.116 15.717 SD 3.565 5.298 5.2293.586 6.154 4.530

The foregoing results indicate that adding elongated and partiallydemineralized bone particles to a calcium phosphate cement did notsignificantly compromise its mechanical properties in the demandingloading modes of shear and bending. In other words, the foregoingresults demonstrate that an osteoconductive, resorbable, andosteoinductive material can be added to a calcium phosphate cementwithout negatively affecting its mechanical properties. In this regard,and without wishing to be bound by any particular theory, it is thoughtthat the results described above further provide support for theproposition that the biologic behavior (incorporation, resorption, newbone formation) of bone cement compositions can be improved by thepresence of the bone materials, without sacrificing the mechanicalproperties of the cement.

Example 14 Analysis of Simple Included Shapes with Two-Dimensional (2D)Connectivity

To further analyze the benefits that may be derived from producingspecially-shaped bone particle and including those particles withincement-based bone graft composition to increase the incorporation,resorption, and remodeling and new bone formation within a resultingcombined hardened material, an analysis was conducted to examine themaximization of the interconnectedness of the included particles, pores,or channels in conjunction with the minimization of the total volume ofincluded particles, pores, or channels. The efficiency with which aparticular shape can achieve complete connectivity is important to theefficacy of that shape compared to other shapes. In other words, whencomparing different shapes of particles for inclusion in the bone graftcompositions of the presently-disclosed subject matter, the shape thatis most connected with the least volume of particle material can, insome instances, be considered most advantageous. Percolation theoryindicates that for a regularly spaced matrix of potential voids, theprobability that a given void exists ranges from 0 to 1 (no voidspresent to all voids present). Furthermore, there will be an importantthreshold probability at which it is possible or probable that acontinuous pathway between voids exists that connects the outer boundaryof the structure to the center of the structure. The level of thethreshold probability depends on the shape of the voids (or in thiscase, the shape of the included particles) and the shape of the overallstructure. See, e.g., Genin D. Percolation: Theory and Applications.NIST. 2007, which is incorporated herein by reference in its entirety.

To conduct the 2D analysis of the shapes of the bone particles, theoverall structure was first represented by a regularly shaped mass ofhardened synthetic bone substitute cement of similar dimensions to afilled defect (2.5 cm×2.5 cm). The cement material was then infused withspecially-shaped bone particles that accelerate the incorporation andremodeling of the synthetic material. Three different shapes wereconsidered. First, circles (spheres in 3D) were used to represent themost general shape indicative of simple grinding of bone in a mill.Second, rectangles were used to represent elongated particles producedthrough a simple machining process. Third, bone shapes in the form of adumbbell having enlarged ends (see, e.g., FIG. 1A) were used as thoseshapes have been shown to have strength benefits, and this analysis wasalso, at least in part, designed to demonstrate any biologic benefits ofthose shapes due to connectivity.

The analysis then used a 10×10 grid space filled with 100 rectangularspaces. In turn, each location could be occupied by a particular shape(First circles, etc.). As the probability of a void presence increased,the number of shapes present then approached 100. A random numbergenerator was used to determine the next location for placement of ashape. Each shape analysis was repeated ten times, and the probabilityvalue at which edge to center connectivity was achieved was recorded.The rectangles and specially-shaped bone particles were able to“connect” (i.e., contact one another) on sides or corners, but thecircles could only “connect” on sides (FIG. 18). The grid wassubsequently randomly filled ten times with corner connectivity, and tentimes with only side connectivity allowed. The resulting thresholdprobabilities were 26.3±7.0% and 46.6±4.6% for corner connectivity(Rectangles and Dumbbells) and side connectivity (Circles),respectively.

TABLE 14 Grid experiment representing a case of side only connectivitythat achieved edge-to-center connectivity at a void probability of 49%.The side-to-side connected pathway reaches one of four center grids(#46) and connects to outer edge grids (#s 8, 9, 30, 40). The variousgrid positions from 1 to 100 were highlighted randomly by sequentialrandom number generation. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1819 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 4243 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 6667 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 9091 92 93 94 95 96 97 98 99 100

TABLE 15 Grid experiment representing a case of side and/or cornerconnectivity that achieved edge-to-center connectivity at a voidprobability of 29%. The connected pathway reaches one of four centergrids (#55) and connects to outer edge grid (#93). The various gridpositions from 1 to 100 were highlighted randomly by sequential randomnumber generation. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2122 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 4546 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 6970 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 9394 95 96 97 98 99 100

Upon examination of the results from this analysis (see Table 16), itwas observed that the volume fraction (area fraction in 2D) of each gridspace occupied by the three shapes are Rectangle: 100%, Dumbbell: 66.7%,Circle: 78.5%. Thus, the amounts of included bone material (volumefraction) of each shape required to achieve threshold connectivity wasfurther calculated to 26.3% for Rectangle shapes, 26.3×0.667=17.5% forDumbbells, and 46.6×0.785=36.6% for Circles. More particularly, it wasfound that, compared to circles, Dumbbell shapes only requireapproximately half the material to achieve comparable connectivity,which is in addition to the strength benefits outline above.

TABLE 16 Result of Various Trials of 2D Connectivity Analysis TrialSquare Circle 1 23 49 2 22 43 3 27 46 4 27 49 5 41 51 6 34 37 7 17 42 829 50 9 22 48 10 21 51 Mean 26.3 46.6 SD 7.04036 4.599517

Example 15 Analysis of Simple Included Shapes with Three-Dimensional(3D) Connectivity

To further assess the connectivity of various shapes, theabove-described analysis was repeated as a three-dimensional analysis bycomparing spheres and dumbbell shapes in a three-dimensional gridpattern similar to that described in the previous example with thefollowing exception: the pattern contained 1000 cube-shaped spaces thatcould be occupied by spheres or by cubes or by dumbbells. The spherescould “connect” only on faces while the cubes and dumbbell shapes couldconnect on faces, edges, and corners. In this regard, the potentialconnectivity of a given space occupied by a cube or a dumbbell shapewith its 26 neighbors is complete with 6 faces, 12 edges, and 8 cornersconnected. With a sphere, there were only 6 potential connections (i.e.,six points of contact).

TABLE 17 Results of Various Trials of 3D Connectivity Analysis. TrialCube Sphere 1 57 305 2 63 297 3 53 383 4 123 344 5 70 241 6 133 257 7 87271 8 155 198 9 119 338 10 76 253 Mean 93.6 288.7 SD 36.0037 55.58787

Upon examination of the results from this analysis, it was found thatthe volume fraction of each grid space occupied by the three shapes areCube: 100%, Dumbbell: 55.5%, Sphere: 52.3%, and thus, the amounts ofincluded bone material (volume fraction) of each shape required toachieve threshold connectivity are 9.4% for Cube shapes, 9.36×0.555=5.2%for Dumbbells, and 28.9×0.523=15.1% for Spheres. More particularly, itwas found that, compared to spheres, dumbbell shapes only require onethird the material to achieve comparable connectivity, which is inaddition to the strength benefits outline above.

Example 16 Finite Element Modeling of Dumbbell-Shaped Bone Particles inCalcium Phosphate Cement

To determine the mechanical behavior effects of adding specially-shaped,processed cortical bone particles to an exemplar calcium phosphatecement loaded in uniaxial tension, a finite element modeling system wasused to model the mechanical properties of dumbbell-shaped particles ascompared to more common shapes, such as simple cylinders. Withoutwishing to be bound by any particular theory, it was thought that if theinterconnecting bone particles demonstrated a mechanical benefitcompared to more common shapes, such as cylinders or spheres, then theunique shapes would be capable of exhibiting a synergistic effect thatcould be achieved in terms of both mechanics and biology.

Briefly, to examine the potential mechanical benefits, finite elementanalyses were performed using an academic research version of acommercially available software package (Ansys Release 12.0.1) runningon a standard PC. The first analysis was set up as a two-dimensionalplane strain simulation using 8 node quadrilateral elements. The testplane was a rectangular region 3.5 mm wide and 4 mm tall. The boundaryconditions were such that the left boundary was fixed in the x-direction(horizontal). The lower left corner was pinned to not move in they-direction (vertical). A uniform x-displacement of 0.0005 mm wasapplied to the right boundary.

Five models (FIG. 19) were created and executed, with the output beingthe Von Mises stress distribution over the test plane. Model 1 wasentirely calcium phosphate cement, Models 2 and 3 contained threerectangular bone particles, and Models 4 and 5 contained threedumbbell-shaped bone particles. In models 3 and 5, the bone pieces had auniform 0.1 mm thick collagen layer around their periphery. The dumbbellparticles that were modeled in these analyses were 2.5 mm long and 1.5mm wide by 0.5 mm thick. Further, in the analyses, the models were 4 mmin height and 3 mm in width.

The calcium phosphate cement was modeled as having the followingmechanical properties: an elastic modulus of 40 GPa; a poisson ratio of0.33; a yield strength of 5 MPa; and a plastic modulus of 5% of theelastic modulus.

The cortical bone was modeled as having the following mechanicalproperties: an elastic modulus of 15 GPa; a poisson ratio of 0.3; ayield strength of 150 MPa; and a plastic modulus of 5% of the elasticmodulus.

The collagen layer (i.e., the demineralized surface) of the corticalbone was modeled as having the following mechanical properties: anelastic modulus of 150 MPa and a poisson ratio of 0.45.

Upon reviewing the results from the finite elements modeling, it wasobserved that the model made up of only calcium phosphate cement (Model1) showed a uniform stress distribution of over 5 MPa indicatingcomplete failure of the cement substance at this level of strain (FIG.20). In Model 2, the inclusion of bone cylinders was somewhat protectiveof the weaker, more brittle calcium phosphate cement (FIG. 21). Therewas, however, a wide path from the top to bottom of the sample thatindicated cement failure and, as such, it was predicted that that samplewould likely have failed completely. In Model 3, the 0.1 mm thickdemineralized layer was of little benefit as the regions of high calciumphosphate cement stress were similar to Model 2 (FIG. 22). Model 4showed a distinct protective effect, with the overlappingdumbbell-shaped particles allowing only isolated regions of high calciumphosphate cement stress (FIG. 23). Finally, Model 5 showed an evenfurther protective effect when the overlapping dumbbell-shaped particleswith a uniform 0.1 mm thick demineralized layer (FIG. 24) were included,as there were only a limited number of small regions of calciumphosphate cement stress in excess of its strength.

In further analyzing the results, it was observed that if the resultswere quantified by calculating the amount of area of calcium phosphatecement that was stressed in excess of its strength (>5 MPa), then theprotective effect of the included bone particles could clearly be seen(FIG. 25). Furthermore, in addition to the obvious mechanical benefitsof the dumbbell shapes, there was less total bone material in Models 4and 5 (6.75 mm²) compared to Models 2 and 3 (7.5 mm²) and there was moretotal length of demineralized (collagen) layer (28.8 mm v. 19.8 mm).Without wishing to be bound by any particular theory, it was thusbelieved that the dumbbell shapes would also be expected to possessbetter biological properties because of the greater amount ofosteoconductive surface that is exposed.

The foregoing results from the finite element modeling represented amechanical benefit from adding the dumbbell-shaped bone particles to thecalcium phosphate cement. The less stiff but stronger dumbbell-shapedbone particles protected the cement from reaching maximum stress. Thebranching parts of the dumbbell-shaped bone particles allowed betterinterdigitation with the cement which aided in the mechanical benefit,especially compared to the straight bars of bone used in models 2 and 3.Unexpectedly, adding the demineralized (collagen) layer to the bonepieces improved the mechanical benefit of adding the dumbbell-shapedbone particles and that unique combination created a composite structurethat could undergo greater strain while minimizing the amount of itscomponent materials reaching critical stress.

Example 17 Bone Volume Handling Test

The specially-shaped bone particles of the presently-disclosed subjectmatter can be incorporated into any bone void filler (BVF), such as acalcium-based cement, and it has been observed that there is an optimumpercent volume of specially-shaped bone particles that should beincorporated in a hardened BVF to achieve a maximum flexural, tensile orcompressive strength. Nevertheless, it was further recognized that therewere other applications in which mechanical strength was of littleconcern, where the needs of a specific biological response and/orremodeling of a bone defect or void was of greater importance than anymechanical strength of the BVF. For example, some applications mayrequire a greater quantity of specially-shaped bone particles, with asmaller percentage of the bone graft compositions volume being taken upby calcium phosphate cement material such that the bone graftcompositions would achieve a faster incorporation and remodeling of adefect space. In this regard, experiments were undertaken to determinehow high of a percentage of the BVF volume could be taken up by thespecially-shaped bone particles before the bone graft composition'shandling characteristics would prevent the material from setting into asolid mass.

Briefly, in these experiments, a calcium phosphate cement consisting oftetracalcium phosphate (TTCP) powder, monocalcium phosphate (MCP)powder, and calcium carbonate powder was used. When these powders weremixed with water in a 2.6:1 powder-to-water ratio by weight, a pasteformed that set within fifteen minutes into a solid mass. Also, in theseexperiments and similar to the methodology described above, corticalbone particles were created through a machining process that yieldedspecially-shaped particles approximately 2.5 mm in length, 1.5 mm inwidth at each end, and 0.5 mm thickness on average with a 0.5 mm widecenter portion (see FIG. 1A). These bone particles were obtained fromthe diaphyseal regions of porcine femora and tibae and were partiallydemineralized using the methodology described above.

After the particles were formed and dehydrated, they were then weighedand added to the premixed cement powder in an approximate 50% volumeratio. The dry components of cement powders and dry bone particles werethoroughly mixed, with the liquid being added to the dry components andmixed for 40 seconds to create a homogenous paste. The wetted and mixedmaterial was very granular and would hold together if compressed. Thematerial was then loaded into two Teflon® (Du Pont de Nemours andCompany Corporation, Wilmington, Del.) cylindrical molds. The molds weredesigned to create test samples 20 mm in length with a diameter of 8 mm.A 7 mm rod was used to compress the material together and into the voidspace of the mold. At 20 min the cylindrical specimens were removed fromthe mold. One specimen was immediately placed in PBS and the other leftin open air to complete setting. At 35 min, the sample in PBS had brokenapart slightly but was hard and set. The sample left to air dry was intwo pieces and also set.

The results from these experiments indicated that adding thesespecially-shaped and partially demineralized bone particles to a calciumphosphate cement at a concentration of 50% bone particles by volumeallows the material to be handled and placed into a defect, even thoughthe material is more wet granules than paste. In other words, theforegoing results demonstrate that an osteoconductive, remodelable,resorbable, and osteoinductive material can be added to a calciumphosphate cement in concentrations on the order of 50% specially-shaped,processed bone particle and still maintain the ability of the bone graftcomposition to set into a hardened mass.

Example 18 Injectability of Bone Graft Compositions

To assess the handling characteristics of a bone graft composition ofthe presently-disclosed subject matter, a calcium phosphate cement withdemineralized, specially-shaped cortical bone particles was investigatedto determine if the handling characteristics of the wetted bone graftcomposition would lend itself to being injected into a defect sight.Briefly, a calcium phosphate cement consisting of tetracalcium phosphate(TTCP) powder, monocalcium phosphate (MCP) powder, calcium carbonatepowder was used. When these powders were mixed with water in a 2.5:1powder-to-water ratio by weight, a paste formed that set within fifteenminutes into a solid mass.

Similar to the methodology described above, cortical bone particles werethen created through a machining process that yielded specially-shapedparticles approximately 2.5 mm in length, 1.5 mm in width at each end,and 0.5 mm thickness on average with a 0.5 mm wide center portion (seeFIG. 1A). These bone particles were obtained from the diaphyseal regionsof porcine femora and tibae and were partially demineralized using themethodology described above. After the particles were formed anddehydrated, the particles were then weighed and added to the premixedcement powder in an approximate 10% volume ratio. The dry components ofcement powders and dry bone particles were thoroughly mixed, with theliquid being added to the dry components and mixed for 30 seconds tocreate a homogenous paste. This wetted material was then loaded into asyringe-style delivery device and injected through a 6 gauge cannula(approx. 4.6 mm ID). Approximately 1 cc was injected into a smallcontainer and covered with Phosphate Buffered Saline (PBS) after 20 minhad elapsed. Another volume of approximately 1 cc was left in open airand allowed to set on the bench top. Both volumes set within 15 min. Theportion that was covered in PBS and left overnight did not show anycloudiness of the solution or signs that the cement material had not setor dissolved to any degree.

The results from these experiments thus indicated that adding thespecially-shaped and partially demineralized bone particles to a calciumphosphate cement did not reduce the ability of the bone graftcomposition to be injected. In other words, the foregoing resultsdemonstrated that an osteoconductive, remodelable, resorbable, andosteoinductive material can be added to a calcium phosphate cement whilealso maintaining the cement bone void fillers ability to be injectedthrough a cannula.

Example 19 Analysis of Hydroxyapatite Cement Augmented with XenograftBone Particles to Improve Incorporation into Cancellous Defects

Hydroxyapatite cement (HAC) is biocompatible and osteoconductive, butits slow resorption limits new bone formation. The addition of pores orbiological materials helps resorption, but typically compromisesshort-term strength. To that end, the effects of adding decalcifiedxenograft bone on cement resorption, new bone formation, and strengthwere determined in an established animal model over ten weeks in orderto examine whether processed xenograft bone particles would increase theincorporation and formation of new bone within HAC without decreasingits strength.

All procedures were approved by an institutional animal care and usecommittee. Briefly, in the first experiment, twelve six-month-old femaleNew Zealand white rabbits were used to compare the biologicincorporation of xenograft versus allograft in a cancellous bone defectin the lateral femoral condyles. Drill-hole defects (8.0 mm long, 4.0 mmdiameter) were prepared and filled with either allograft cancellous boneobtained from other rabbits or xenograft cancellous bone from youngpigs. The bone graft was washed in organic detergent (Triton X-100) andmorselized to an average particle size of 2 mm. Graft samples wereimpacted in a tube before being placed in the defect. Histologicanalysis was done on femur samples after three (n=6) or ten (n=6) weeks.The amount of new bone was measured, and the degree of inflammatoryresponse was evaluated and quantified using a 0-3 analog scale. Calceinwas given three days before sacrifice to indicate newly forming bone.

In the second experiment, eight six-month-old female New Zealand whiterabbits were used. Drill-hole defects (8.0 mm long, 5.0 mm diameter)were again prepared, but were filled with either HAC alone or HAC mixedwith processed xenograft bone particles from young pigs (XBC) at avolumetric ratio of approximately 25%. The particles were elongated“needles” of cortical bone that were approximately 5 mm long and had adiameter of approximately 1 mm. The cortical bone particles wereextensively washed, demineralized in dilute HCl, and rewashed.Micro-Computerized Tomography (μCT) scanning, decalcified andundecalcified histology, and mechanical indentation testing of thehealing defects were performed after ten weeks (n=8). New bone andinflammatory/immune response were graded on a 0-3 scale, and calceinlabeling was quantified as the percent area of new bone. Statisticalanalysis was by Student's t-tests.

Subsequent to the analysis of the results from the first experiment, itwas observed that the allograft cancellous bone healing response showeda rapid incorporation of the graft with extensive new bone formation at3 weeks and nearly complete remodeling at 10 weeks (FIG. 26A) with aminimal inflammatory response and new bone formation (FIG. 26B). In manycases it was difficult to distinguish the allograft defects from normalbone at 10 weeks. The xenograft cancellous bone healing response wasconsiderably slower (FIG. 26C) with a much larger inflammatory responseat both 3 and 10 weeks and a generally higher level of new boneformation (FIG. 26D). By ten weeks, there was new bone formation in bothgroups, but with substantial new bone with the xenograft (FIG. 27A). Thedecrease in bone in the 10-week allograft group was because of extensiveremodeling back to the normal state. Again, the inflammatory responsewas greater with xenograft and remained greater over 10 weeks (FIG.27B).

Subsequent to the analysis of the results from the second experiment, itwas observed that, overall, the XBC group showed significantly more newbone formation than the HAC group throughout the defect (p<0.05). TheXBC group, however, showed significantly more inflammatory/immuneresponse than the HAC group (p<0.05). The three-dimensional μCTreconstructions showed that the HAC was basically inert, while the XBCtook on an appearance suggestive of more extensive incorporation.Indeed, after 10 weeks, the HAC cylinder (FIG. 28A) was well fixed andappeared to be integrated with the cancellous bone, but without muchremodeling, while the XBC cylinder (FIG. 28B) appeared to be remodelingmore extensively with new bone formation. The indentation strength ofHAC was significantly stronger than XBC only after ten weeks (p<0.05),but both grafts were stronger than normal cancellous bone at all times.

In the second experiment, after 10 weeks, there was no new boneformation within the HAC filled defect (FIG. 29A), while the XBC filleddefect (FIG. 29B) had several regions of extensive cellular activitywith new bone formation (see, e.g., FIG. 29C). After 10 weeks, there wasalso significantly more new bone formation within the XBC defectscompared to HAC (FIG. 30A; p<0.05). There was also significantly moreinflammatory/cellular response in the XBC compared to HAC (FIG. 30B;p<0.05). Further, after 10 weeks, there was a trend toward more new boneformation activity within the XBC defects (FIG. 31A), as indicated bycalcein labeling, compared to HAC (FIG. 31B) (see also FIG. 31C).Finally, blunt indentation testing using a 1.5 mm diameter indentershowed no significant difference at time zero, but the XBC resistedsignificantly less indentation force after 10 weeks in vivo (FIG. 32).It should be noted that the XBC remained stronger than cancellous boneafter 10 weeks.

The results from the foregoing experiments indicated that addingxenograft to HAC created a bioactive composite that was more rapidlyincorporated, resorbed, and replaced by new bone. The presence of thexenograft particles created a vigorous inflammatory response, but,without wishing to be bound by any particular theory, it was thoughtthat there may be some benefit to the resorption rate of the HACcomponent of the XBC due to the infiltration of cells. That volumetricinclusion of rapidly resorbed bone graft did not compromise the initialindentation strength of the filled defect relative to normal cancellousbone.

Example 20 Allograft Augmented Calcium Phosphate Cement as aSelf-Hardening Bone Graft Substitute

An established animal model was utilized to determine the effects ofadding specially-engineered shapes of partially demineralized allograftcortical bone on short term cement behavior and biocompatibility, and tofurther assess whether the presence of processed bone particles wouldincrease the incorporation rate of the cement without compromising itshandling characteristics. Briefly, a six-month-old female New Zealandwhite rabbit was used to evaluate the handling characteristics andbiologic incorporation of allograft augmented calcium phosphate cementin cancellous bone defects in the lateral femoral condyles of both hindlimbs. All procedures were first approved by the institutional animalcare and use committee. First, drill-hole defects (8.0 mm long, 5.0 mmdiameter) were prepared and filled with a novel calcium phosphate cementaugmented with specially engineered shapes of partially demineralizedallograft cortical bone. The particles were elongated “dumbbells” ofcortical bone approximately 2.5 mm long, 1.5 mm diameter at the ends and0.5 mm diameter in the center portion (see FIG. 1A). Prior to mixingwith the cement, the particles were machined from a donor rabbit femur,extensively washed, demineralized in dilute HCl, and rewashed. The outerlayer of each dumbbell was demineralized to a thickness of approximately100 to 200 μm. The cleaned particles were allowed to dry in air in anoven at 37° C. for 24 hours and were then sealed in sterile airtightcontainers prior to use.

At the time of surgery, and subsequent to the creation of the defect,the cement was mixed by combining 2.4 g of calcium phosphate cementpowder and thirty dumbbell-shaped particles (see FIG. 1A) of partiallydemineralized bone (0.064 g). The partially demineralized bone particleswere first added to the container of calcium phosphate cement powder andmixed thoroughly with a spatula to evenly disperse the bone within thepowder. Next, a sodium phosphate solution was added to the dry mixtureof calcium phosphate cement and partially demineralized bone particles.The material was then mixed for 30 sec to form a paste with a uniformconsistency. Once a uniform consistency was obtained, a spatula was thenused to fill the drill hole defect that was created in the distal femurof the rabbit. As a result of filling the defect with the paste, eachvoid was made to contain approximately 9 to 12 pieces of demineralizedbone, or, in other words, about 10 to about 15% demineralized boneparticles by volume of the defect. The outer surface of the defect wasthen covered with a plug of bone wax to stop bleeding and to preventperiosteal osteogenesis in the defect. The wound was then closed and theprocess was repeated for the opposite leg.

The rabbit was subsequently allowed to recover and ambulate normally andeat a normal diet ad libitum. After ten days, the rabbit was sacrificed,and high resolution μCT scanning (14 μm voxels) was performed on bothdistal femurs before they were prepared for decalcified histology usinghemotoxylin and eosin staining.

Upon obtaining the results of these experiments, the micro-CT scan image(FIG. 33) shows the extent to which the experimental bone substitutecement was able to fill the defect and the extent to which the hardenedcement extends and the presence of several partially demineralizedallograft “dumbbells.” Also, the allograft dumbbells are clearly visibleat several locations within the filled defect (see, e.g., FIG. 34), andthe demineralized layer on the bone particles is clearly visible due tothe radiolucency of that layer. Further, other allograft bone piecescould be seen at other levels in different regions of the defect.

The histology from the above-described experiments showed the extent towhich the experimental cement filled the defect (FIG. 35), and clearlyshowed that there was a distribution of allograft bone pieces throughoutthe cross-section. A single “dumbbell” was seen in a horizontalorientation and others are at various orientations outside the plane ofthe slice. At a higher magnification (100× magnification) of thehistology sections, cellular infiltration at the boundary of the defectinto the demineralized layer of the allograft bone piece was observed(FIG. 36), and the incorporation of an allograft particle from thedefect boundary via the demineralized layer was also observed along witha faint mark between the demineralized bone layer and the mineralizedbone (FIG. 37). Additional histological sections showed the cellularinfiltration and incorporation of allograft particles with a faint markbetween the demineralized bone layer and the mineralized bone (FIG. 38).

A 3-D micro-CT reconstruction (FIG. 39A) of the distal femur regioncontaining the filled drill-hole defect was further used to examine theability of the bone graft composition to treat the drill hole defect. Inthis regard, a transverse trim of the reconstruction through the middleof the defect showed the extent to which the hardened cement extendedthrough the entire region of the defect and the presence of severalpartially demineralized allograft “dumbbells” within the defect itself(FIG. 39B). When the reconstruction of the defect was trimmed from thetop and front, the allograft bone pieces could be seen at other levelsin different regions of the defect (FIG. 39C). Also, the demineralizedlayer on the bone pieces was clearly visible due to its radiolucency.

To further examine the bone graft composition, an approximately 2 mmthick slab reconstruction of the cement filled defect region wascreated. That reconstruction showed cross-sections of several corticalallograft “dumbbells” within the cement-filled defect region (FIG. 40A).Additionally, the same slab, viewed with a different threshold, showedthe collagen (demineralized) layer on the allograft “dumbbells” (FIG.40B). Regions outside the defect region were also highlighted becausethey have the same CT density as the collagen. In both reconstructions,however, the included dumbbell-shaped bone particles were interconnectedand were distributed through the defect giving a trabecular-likeappearance.

A 0.5 mm thick slab reconstruction of the cement filled defect regionfurther showed cross-sections of at least two cortical allograftdumbbells (FIG. 41A). The same slab with a different threshold alsoshowed the collagen (demineralized) layer on the allograftdumbbell-shaped particles (FIG. 41B). Regions outside the defect regionwere also highlighted because they had the same CT density as thecollagen. Similar to the reconstructions discussed above, the 0.5 mmslab also revealed dumbbell-shaped particles that were interconnectedand were distributed throughout the defect, giving a trabecular-likeappearance.

The foregoing results demonstrated that a bone substitute cementcomprised of calcium phosphate cement and dumbbell-shaped cortical boneparticles could be handled and delivered similar to existing bonesubstitute cements. Further, the distribution of the specially processedallograft bone particles was evident from the histology sections and,where the particles interacted with the host tissue at the defectboundaries, there was a rapid infiltration of cells and evidence ofresorption of the graft and calcium phosphate cement. Healing and newbone formation was also apparent in some locations after only 10 days.

The dumbbell-shaped bone particles also appeared to improve thedistribution of the bone graft and to increase the likelihood of contactwith the defect boundaries. Additionally, it appeared that thedemineralized layer facilitated the resorption and infiltration of thebone substitute and promoted incorporation and new bone formation afterten days in vivo.

Example 21 Analysis of a Bone-Augmented Calcium Phosphate Cement as aSelf-Hardening Bone Graft Substitute in a Rabbit Model

To determine the effects of adding specially-shaped, partiallydemineralized human cortical bone on short-term cement behavior andbiocompatibility, and to further assess whether the presence of thoseprocessed bone particles increases the incorporation rate of the cementwithout compromising its handling characteristics, an established animalmodel is utilized. Briefly, 36 six-month-old female New Zealand whiterabbits are used to evaluate the handling characteristics and biologicincorporation of allograft-augmented calcium phosphate cement incancellous bone defects in the lateral femoral condyles of both hindlimbs. All procedures are approved by an institutional animal care anduse committee. First, drill-hole defects (8.0 mm long, 5.0 mm diameter)are prepared and filled with a bone graft composition of thepresently-disclosed subject matter comprising a calcium phosphate cementaugmented with specially-shaped, partially demineralized allograftcortical bone. The particles are elongated dumbbell-shaped pieces ofcortical bone approximately 2.5 mm long, 1.5 mm wide at the ends, andhave a 0.5 mm thickness in the center portion. Prior to mixing with thecement, the cortical particles are machined, extensively washed,demineralized in dilute HCl, and rewashed, lyophilized, and sterilized.The outer layer of each processed bone particle is then demineralized toa thickness of approximately 50 to 200 μm.

At the time of surgery, and subsequent to the creation of the defect,the sterile cement powder is mixed by combining calcium phosphate cementpowder and processed particles of partially demineralized bone. Thepartially demineralized bone is first added to the container of calciumphosphate cement powder and mixed thoroughly with a spatula to evenlydisperse the bone within the powder. Next, a sodium phosphate solutionis added to the dry mixture of calcium phosphate cement and partiallydemineralized bone. The material is then mixed for 30 sec to form apaste with a uniform consistency. Once a uniform consistency isobtained, a spatula is then used to fill the drill hole defect createdin the distal femur of the rabbit. As a result of filling the defectwith the paste, each void is made to contain about 10 percent to about15 percent demineralized bone structures by volume of the defect. Thewound is then closed and the process is repeated for the opposite leg.

The rabbits are subsequently allowed to recover and ambulate normallyand eat a normal diet ad libitum. After three weeks (n=12), 8 weeks(n=6), 12 weeks (n=12), and 24 weeks (n=6), the rabbits are sacrificedand high resolution μCT scanning (14 μm voxels) is performed on bothdistal femurs before they are prepared for decalcified and/orundecalcified histology. Some samples are separately prepared formechanical testing to measure the compressive strength of the repaireddefect together with surrounding bone at various time points.

At each time point, the histology reveals that there is more new boneformation within the treated defects containing the partiallydemineralized bone. Analysis of the mechanical behavior of the bone anddefect also reveals that the strength of the repair is maintained at orabove the baseline strength of representative cancellous bone during theexperimental timeframe. Together, these results demonstrate that a bonegraft composition of the presently-disclosed subject matter comprising acalcium phosphate cement augmented with specially-engineered shapes ofpartially demineralized allograft cortical bone, can effectively be usedas part of a method for treating a bone defect.

Example 22 Osteoinductivity of a Calcium Phosphate Based CompositeCement Containing Processed Cortical Bone Particles

Assessment of ectopic bone formation in an athymic nude rat model afterimplantation in intramuscular or subcutaneous pockets is the currentstandard for assessing the osteoinductive properties of implantablematerials. As such, an athymic nude rat model is thus utilized in anexperiment to assess the osteoinductive properties of the bone graftcompositions of the presently-disclosed subject matter and to comparethose compositions to other materials. Briefly, test bone graftcompositions are prepared by combining a calcium phosphate powder, asetting solution, and partially demineralized, specially-shaped, boneparticles. The test samples are then formed into a 5 mm disc with a 0.5mm thickness, and the discs are bilaterally implanted in four (4)animals in subcutaneous muscle pouches in the axiliary region of eachthe nude athymic male rats. Once anesthetized, a small incision is madeand an opening is created through the skin, subcutaneous tissue, andfascia of each animal. A muscle pouch is then formed by cutting in thesame direction as the muscle fibers, and the scissors are then used andspread to create a pouch in the muscle. The 0.1 ml test sample is thencarefully inserted using forceps and, once the test article is verifiedto be properly in place and maintaining form, the muscle pocket issutured closed. The surgery is then repeated in the bilateral implantsite. If necessary an additional half dose of ketamine/xylazine isadministered to maintain a level of anesthetization sufficient tocomplete the implantation procedure.

Implant sites are harvested en bloc after 28 days post-implantation andsent for histological preparation. The histological slides are scoredfor osteoinductivity and inflammatory responses are recorded inaccordance with the scoring system of Edwards et al. (1998), which makesuse of a 0-4 scale, where 0 indicates no evidence of bone formation, and1, 2, 3, and 4 indicate less than 25%, 26-50%, 51-75%, and greater than75%, respectively, of implant surface involved in new bone formation. Inaddition, bone maturity is scored in accordance with Katz et al. (2006).During the scoring, each of the samples are randomized and blinded tothe investigators.

Upon analysis of the scoring, it is observed that, on average, theimplanted test disks incorporating the processed bone reinforcingelements produces a valid osteoinductive score compared to the controlsamples with 100% cement, indicating that adding specially-shaped andpartially demineralized bone particles to a calcium phosphate cementimprove its osteoinductive properties, and further indicating that boththe biologic behavior (incorporation, remodeling, new bone formation)and the mechanical behavior of bone cement compositions can be improvedby the presence of the specially-shaped bone materials.

Example 23 Aqueous Buffering of the Cement Powder, Processed BoneParticles, and Setting Liquid Combination

The presence of the specially-processed bone particles of thepresently-disclosed subject matter allows some flexibility in thehandling of the ratios of powder to liquid when preparing cement pastes.Further, the presence of the bone particles with a demineralizedcomponent or additional porosity allows for water to be absorbed orreleased by the bone particles in a way that positively affects thehandling and/or strength characteristics of the cement. Moreover, thespecially-shaped bone particles have a decalcified layer that gives theparticles the ability to absorb small amounts of moisture. Each of thesecharacteristics was considered to be important as the mass of water orother biological fluid that the specially-shaped bone particles can holdcan be considered in light of the amounts of osteogenic proteins, water,blood, antibiotics, and other such materials that may be held by theparticles. In this regard, it is additionally thought that the residualmoisture the specially-shaped bone particles can absorb may also be usedto alter the handling characteristics of calcium salt bone void fillers.

Thus, to more precisely determine the amount of moisture the individual,specially-shaped bone particles are capable of retaining, five separatebatches of a known amount of specially-shaped bone particles were firstdehydrated at 40-42° C. with convection for four hours and then eachbatch was weighed. This mass was recorded as the batch ofspecially-shaped bone particles dehydrated weight. The batch ofparticles was then super-hydrated by covering the particles withdeionized water and leaving the particles under vacuum for one hour.After hydrating the particles, each particle was removed from the waterbath and the residual moisture on the surface of the individualparticles was removed with a lint-free cloth by briefly blotting thesurface. Once the surface moisture was removed, the particles were thenre-weighed, and that subsequent weight was considered the fully-hydratedweight. The difference in mass from the dehydrated to the fully-hydratedstate was then used to calculate the mass of water. Additionally, tofurther confirm the mass of the dehydrated bone particles, the particleswere then placed back in the dehydrator and left under convection at40-42° C. until the initial mass of the batches plateaued. The batcheswere weighed at multiple time points. Initially the mass decreasedrapidly in the first few time points, with the difference in the mass ofthe batches between time points then decreasing gradually as the elapsedtime increased.

TABLE 18 Differential Weights of Hydrated and Dehydrated Bone ShapesWeight Fully Redried Group of Tr Dehydrated Hydrated Difference % 5 min15 min 40 min 1 1.5324 17.9535 18.5824 0.6289 41.0402 18.3544 18.162217.9999 2 1.532 18.0937 18.6642 0.5705 37.2389 18.4725 18.3288 18.1301 31.5335 17.9426 18.6457 0.7031 45.84936 18.496 18.2766 18.0053 4 1.531717.9541 18.6085 0.6544 42.72377 18.4104 18.2049 18.0077 5 1.5497 18.187118.8293 0.6422 41.44028 18.7086 18.5136 18.255 Mean 41.6585 SD 3.108894

Upon analysis of the results from these experiments, and as shown inTable 17 above, it was determined that the specially-shaped andprocessed, partially demineralized cortical bone particles (Tr) canretain approximately 40% of their mass in water. At the ratios of powderto liquid typical employed in the foregoing in vivo experiments, thisamount of water represented a significant variation in the amount ofliquid that could be mixed with the powder to form a workable cementpaste.

Example 24 Autologous Factor Carrying Capacity of the Included BoneParticles and Mechanical Effects of Including Autologous Factors

As noted, the presence of the specially-shaped bone particles allowsadditional flexibility in the handling of the ratios of powder to liquidwhen preparing cement pastes. This property, in turn, is believed tofurther allow blood (or other autologous factors) from a subject to beused to precondition the included bone particles for better biologicalbehavior in vivo. Further, the presence of the bone particles with ademineralized component or additional porosity is also believed to allowfor autologous factors (blood, serum, cells, bone marrow aspirate, etc.)to be absorbed or adsorbed by the bone particles for biologic benefitsin a way that does not negatively affect the handling and/or strengthcharacteristics of the cement. To assess the extent of these benefits,experiments are performed to determine whether adding a small amount ofblood to dry bone particles, before mixing the particles with cementpowder and setting liquid, would negatively affect the subsequentmechanical properties of the final product.

Briefly, and similar to the experiments described above, mechanicaltesting experiments using calcium phosphate cement with 10% by volumepartially demineralized, specially-shaped cortical bone particles areundertaken to determine the mechanical behavior effects of adding bloodto the specially processed cortical bone particles. In theseexperiments, a calcium phosphate cement consisting of tetracalciumphosphate (TTCP) powder, monocalcium phosphate (MCP) powder, calciumcarbonate powder is used and, similar to the methodology describedabove, cortical bone particles are also created through a machiningprocess that yields specially-shaped particles approximately 2.5 mm inlength, 1.5 mm in width at each end, and 0.5 mm thickness on averagewith a 0.5 mm wide center portion (see FIG. 1A). These bone particlesare again obtained from the diaphyseal regions of porcine femora andtibae and are partially demineralized using the methodology describedabove. After the particles are formed and dehydrated, they are thenweighed to represent a 10% volume ratio of the total mix (as describedabove in previous examples). Before the dry components of cement powdersand bone particles are mixed together, a small quantity (0.25 cc) ofhuman blood is mixed with the bone particles (0.345 g), and these areallowed to soak in a shallow bowl for approximately 2 minutes. Next, thebone particles are removed from the blood and mixed with the dry cementpowder in sufficient quantity to create 3 cc of cement paste.

Test samples are then created by combining the dry materials with anappropriate amount of setting liquid, mixing with a thin metal spatula,until a consistent wet paste is formed. Next, the materials are spreadinto cylindrical Teflon® (Du Pont de Nemours and Company Corporation,Wilmington, Del.) molds. The molds are designed to create test samples20 mm in length with a diameter of 8 mm.

The bending test is then performed using a three-point bending fixtureas described above (see, e.g., FIG. 3). Upon analysis of the resultsfrom these experiments, it is observed that adding blood to thespecially-shaped and partially demineralized bone particles prior tocombining them with a calcium phosphate cement does not significantlyaffect the mechanical properties of the resulting bone graft compositionin the demanding loading mode of bending.

Example 25 Elution Properties of the Combined Cement and Bone Particlesfor Delivery of Therapeutic Agents, Including Antibiotics and GrowthFactor Proteins

To assess the ability of the bone graft compositions of thepresently-disclosed subject matter to be utilized as delivery systemsfor various therapeutic agents, experiments were performed to assess theelution of various agents of interest from the hardened end product whenit is incubated in a liquid environment. Briefly, in these experiments,to test the elution of growth factor proteins such as bone morphogeneticproteins (BMPs), an analog protein lysozyme was used, and to testantibiotic elution, vancomycin was used. A bone graft composition of thepresently-disclosed subject matter that was comprised of calciumphosphate, consisting of tetracalcium phosphate (TTCP) powder anddicalcium phosphate anhydrous (DCPA) powder, was used in theseexperiments plus approximately 25% specially-shaped cortical boneparticles (by volume). Lysozyme (Sigma, St. Louis, Mo.) was pre-adsorbedonto the bone particles before they were added to the cement to achieve0.0168 or 0.168% loading. In this regard, lysozyme was dissolved in 0.85mL of setting solution at a concentration of 0.44 and 4.4 mg/mL and wasthen added to the bone particles. The protein-bone mixtures were thenrotated at room temperature for 30 min, after which the mixture wasadded to 2.225 g of calcium phosphate powder. Vancomycin (Sigma, St.Louis, Mo.) was dry-mixed with the calcium phosphate powder (2.225 g)and bone particles prior to addition of the setting solution (0.85 mL).Either 0.3, 3.0, or 10.0% (6.68, 66.8, or 222.5 mg) of antibiotic wasadded to each sample. Each batch of antibiotic- or protein-loadedcement/bone mixture was spread in a mold to form samples of 19 mmdiameter and 1.5 mm thickness. For comparison, drug-free cement sampleswere made using 2.225 g cement, bone particles, and 0.85 mL settingsolution. After allowing samples to set overnight at room temperature,release experiments were conducted.

For the release experiments, samples were immersed in 3 ml of 150 mMphosphate-buffered saline, pH 7.4, and incubated at 37° C. with gentleshaking. All of the supernatant was collected and replaced daily tomaintain sink conditions and a constant volume. Vancomycin concentrationwas determined by measuring absorbance at 280 nm (Biotek PowerWave HT UVmicroplate reader, Bio Tek Instruments Inc., Winooski, Vt.) andcomparing to a standard curve constructed with the antibiotic. Lysozymeconcentration was measured using the MicroBCA Protein Assay (ThermoFisher, Waltham, Mass.) according to the manufacturer's instructions,with the exception that the samples were incubated at 37° C. for twohours following addition of the working reagent to the supernatant.Absorbance was measured at 570 nm and compared to standard curvesprepared with lysozyme. For both the antibiotic and protein assays,results from drug-free samples were used to correct for nonspecificbiomaterial effects.

Upon analysis of the results from these experiments, it was found thatthe bone graft compositions of the present invention were capable ofreleasing both the growth factor analog protein, lysozyme (FIGS. 42A and42B), and the antibiotic, vancomycin (FIGS. 43A and 43B). The lysozymewas released in similar amount regardless of the amount used because theavailable collagen layers of the bone particles controlled the protein.The vancomycin was released in a dose-dependent manner when placed in aliquid environment because it was mixed throughout the cement. FIGS. 42Aand 43A show the measured amount (μg) of lysozyme or vancomycin presentin the supernatant at each point when it was replaced. The sum total(μg) of the lysozyme or vancomycin released is shown in FIGS. 42B and43B. Due to the in vitro nature of the experiments, there were nodissolution of the hardened cements. Thus, only the antibiotic powder atthe sample surface was available for release into the surrounding fluid.The rising cumulative release of lysozyme or vancomycin illustrates thatthe addition of specially-shaped and partially demineralized boneparticles to a calcium phosphate cement can be utilized as an effectivedelivery systems for various therapeutic agents.

Throughout this document, various references are mentioned. All suchreferences are incorporated herein by reference, including thereferences set forth in the following list:

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It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thesubject matter disclosed herein. Furthermore, the foregoing descriptionis for the purpose of illustration only, and not for the purpose oflimitation.

What is claimed is:
 1. A bone graft composition, comprising: abiologically-resorbable cement; and a plurality of processedinterconnecting bone particles, each of the interconnecting boneparticles being cut from an intact whole bone or portion thereof, eachof the interconnecting bone particles having larger dimensions at itsends compared to its center, and each of the interconnecting boneparticles having a shape configured to interconnect with adjacentinterconnecting bone particles, wherein the bone graft composition isconfigured to form a network of interconnected pathways or channels dueto interconnected adjacent interconnecting bone particles, wherein thenetwork of interconnected pathways or channels are configured to allowfor cells and fluids to infiltrate the bone graft composition.
 2. Thebone graft composition of claim 1, wherein each of the interconnectingbone particles is further configured to interdigitate with thebiologically-resorbable cement.
 3. The bone graft composition of claim1, wherein the biologically-resorbable cement is a calcium-based cement.4. The bone graft composition of claim 3, wherein the calcium-basedcement is a calcium phosphate cement.
 5. The bone graft composition ofclaim 3, wherein the calcium-based cement is a calcium sulfate cement.6. The bone graft composition of claim 1, wherein each of theinterconnecting bone particles includes a plurality of enlarged portionsand a plurality of center portions aligned along a common longitudinalaxis, each of the enlarged portions extending laterally away from acommon longitudinal axis of each center portion, and each of the centerportions interposed between respective enlarged portions.
 7. The bonegraft composition of claim 1, wherein a cross-section of theinterconnecting bone particles is substantially round, elliptical,square, rectangular, or triangular in shape.
 8. The bone graftcomposition of claim 1, wherein the interconnecting bone particles areabout 5% to about 90% demineralized.
 9. The bone graft composition ofclaim 1, wherein the interconnecting bone particles comprise corticalbone, cancellous bone, or both cortical and cancellous bone.
 10. Thebone graft composition of claim 1, wherein the interconnecting boneparticles are selected from the group consisting of autograft boneparticles, allograft bone particles, xenograft bone particles, andcombinations thereof.
 11. The bone graft composition of claim 1, whereinthe interconnecting bone particles comprise about 1 percent to about 50percent by volume of the bone graft composition.
 12. The bone graftcomposition of claim 1, wherein the composition further comprises anosteoinductive material, an osteogenic material, or both.
 13. The bonegraft composition of claim 1, wherein the composition further comprisesan antibiotic.
 14. A kit, comprising: a biologically-resorbable cementpowder; and a plurality of processed interconnecting bone particles,each of the interconnecting bone particles being cut from an intactwhole bone or portion thereof, each of the interconnecting boneparticles having lamer dimensions at its ends compared to its center,and each of the interconnecting bone particles having a shape configuredto interconnect with adjacent interconnecting bone particles, whereinthe plurality of processed interconnecting bone particles, when combinedwith the biologically-resorbable cement powder, are configured to form anetwork of interconnected pathways or channels due to interconnectedadjacent interconnecting bone particles.
 15. The kit of claim 14,wherein the interconnecting bone particles are lyophilized.
 16. The kitof claim 14, further comprising an aqueous vehicle for adding to thebiologically-resorbable cement powder, the interconnecting boneparticles, or both the biologically-resorbable cement powder and theinterconnecting bone particles.
 17. The kit of claim 14, wherein thebiologically-resorbable cement powder is contained in a first vessel,and wherein the processed interconnecting bone particles are containedin a second vessel.
 18. The kit of claim 14, wherein thebiologically-resorbable cement powder and the processed bone particlesare packaged together in a single vessel.
 19. The bone graft compositionof claim 1, wherein the interconnecting bone particles comprisecancellous bone, wherein the cancellous bone comprises collagenoustrabeculae with tunnel-like spaces configured to allow cells and fluidsto infiltrate the bone graft.